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  • Life Sciences
  • 1
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: The increasing requirements in wastewater treatment have led to the development of new wastewater treatment processes based on the know-how and experience in reaction and process engineering of the chemical industry. Due to their compactness, closed operation and high flexibility, these new processes show a large potential for process integration and significant cost reduction in particular for highly polluted industrial wastewaters.This paper discusses the HCR (high-performance compact reactor) - process, developed at the Mass Transfer Laboratory of the Technical University of Clausthal within the last decade. This process has been realized in more than 30 technical applications with a volume loading of up to 70 kg COD/m3 d and an energy consumption of about 0.4 kWh per kg CODelim.
    Additional Material: 10 Ill.
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  • 2
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Berlin : Wiley-Blackwell
    Acta Biotechnologica 20 (2000), S. 169-174 
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Nitrogen fixing Anabaena azollae strains isolated from four different Azolla cultures were characterized based on their total protein profile and RAPD profile to study the existing variation among them. As expected, the isolates showed almost similar protein banding patterns, but exhibited differences in 40-70 KDa protein subunits. Polymerase chain reaction of the DNA of the isolates, using four different primers, amplified specific sequences of DNA and showed clear polymorphism among the isolates. The RAPD profile generated the fingerprinting pattern characteristic of each strain based on the sequence of the primers used. Common band sharing observed between the strains A. azollae-RS-KK-SK-AM and A. azollae-RS-KK-SK-RP probably represents maternal inheritance of DNA to the progeny. The polymorphic bands were generated specifically for the isolates A. azollae-RS-KK-SK-RP and A. azollae-RS-KK-SK-AM with primers numbered 2 and 4, respectively, which could be developed as possible markers for these isolates.
    Additional Material: 1 Ill.
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  • 5
    Electronic Resource
    Electronic Resource
    Berlin : Wiley-Blackwell
    Acta Biotechnologica 20 (2000), S. 175-183 
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Mature seed-derived callus from an elite Chinese japonica rice cv. Eyl 105 was transformed with a plasmid containing the selectable marker hygromycin phosphotransferase (hpt) and the reporter β-glucuronidase (gusA) genes via particle bombardment. After two rounds of selection on hygromycin (30 mg/l)-containing medium, resistant callus was transferred to hygromycin (30 mg/l)-containing regeneration medium for plant regeneration. Twenty-three independent transgenic rice plants were regenerated from 127 bombarded callus with a transformation frequency of 18.1%. All the transgenic plants contained both gusA and hpt genes, revealed by PCR/Southern blot analysis. GUS assay revealed 18 out of 23 plants (78.3%) proliferated on hygromycin-containing medium had GUS expression at various levels. Genetic analysis confirmed Mendelian segregation of transgenes in progeny. From R2 generations with their R1 parent plants showing 3:1 Mendelian segregation, we identified three independent homozygous transgenic rice lines. The homozygous lines were phenotypically normal and fertile compared to the control plants. We demonstrate that homozygous transgenic rice lines can be obtained via particle bombardment-mediated transformation and through genetic analysis-based selection.
    Additional Material: 4 Ill.
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  • 6
    Electronic Resource
    Electronic Resource
    Berlin : Wiley-Blackwell
    Acta Biotechnologica 20 (2000), S. 202-202 
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    Berlin : Wiley-Blackwell
    Acta Biotechnologica 20 (2000), S. 219-233 
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: The GRAM-positive bacterium Rhodococcus erythropolis K2-3 and the GRAM-negative Ochrobactrum anthropi K2-14 are capable of synergistically degrading 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB). The two strais execute this task in a symbiotic manner, but the nature of the interaction involved in the degradation is only partially understood as yet. An essential first step in elucidating the interaction is to be able to monitor the two strans separately, at the cellular level, within mixed populations. Therefore a method exploiting fluorescently labelled lectin probes was developed. Since Concanavalin A (Con A) binds specifically to R. erythropolis K2-3, it was selected and linked to the fluoresent dye Bodipy 630/650, which has an excitation maximum in the red part of the visible light spectrum. Forward light scatter (FSC) and DNA fluorescence from both strains were also measured to obtain simultaneous information about their physiological states. The three parameters were conveniently monitored by dual and triple excitation flow cytometry in conjunction with double fluorescent staining techniques. In addition, the strains were identified using an epifluorescence microscope. These techniques were found powerful tools for the population analysis of this mixed bacterial system.
    Additional Material: 7 Ill.
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  • 8
    Electronic Resource
    Electronic Resource
    Berlin : Wiley-Blackwell
    Acta Biotechnologica 20 (2000), S. 235-274 
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: A quite unconventional, innovative scientific methodology called “macroscopic pattern analysis” is presented in this paper. This approach is more adequate in the case of complex systems than the well-known microscopic, mechanistic approach. Complex systems are not only attracting more engineering interest, but their scientific treatment is increasingly wanted by society due to the manifold problems in Earth's ecosphere. The macroscopic pattern approach will be explained in depth and illustrated in some case studies from the ecosphere (sustainability, hurricanes and avalanches), where nature serves as a teacher for the solution of the sustainability problem. Then, a series of case studies on macropatterns are described showing the problem-solving capacity for anthropo- and technosphere: sustainability in society with an index of sustainability, the eco-social market economy with eco-tech as an instrument, biokinetics, bioreactor mixing and integrated bioprocessing with models, design of cars and houses and even quality of life as an attempt to quantify macropatterns.The innovations are briefly compared in their problem-solving capacity with known approaches such as the microscopic method in science, technology and society (free market economy), including the evaluation of other indices and cleaner production, industrial ecology and zero emission initiative. Finally, a deeper integration of sciences, ethics, arts and nature will be introduced based on the vision with macroscopic pattern analysis, where the different domains of human life are integratable to effect a reconciliation.
    Additional Material: 22 Ill.
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  • 9
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
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  • 10
    Electronic Resource
    Electronic Resource
    Berlin : Wiley-Blackwell
    Acta Biotechnologica 20 (2000), S. 17-29 
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: The dynamic behaviour of biofilters intended to remove toluene and xylene from air was studied during transient states. Laboratory scale biofilters were filled with a mixture of peat, bark and wood and inoculated with a mixed microbial population. Toluene and xylene were applied both as single pollutants and as mixtures. Attention was focused on the evaluation of the following transients: the response of biofilters to step changes and peaks in pollutant concentrations, the effect of changes between single and multiple pollutant loadings and the response to shutdown periods.The biofilters demonstrated a good dynamic stability during transient states induced by change in inlet pollutant concentrations. Their time periods did not exceed three hours. No interaction between xylene and toluene degradation was observed during changes in loading with single pollutants or their mixture. The performance interruptions lasting less than 24 hours were found to have no significant influence on the removal efficiency of biofilters. When the biofilters were reacclimated after longer starvation periods, a short temporary decrease in efficiency whose minimum and duration were proportional to the length of a preceding shutdown period was observed. The longest starvation period (7 days) resulted in a reacclimation lasting 7 hours only. Adaptations of a microbial population to new operating conditions as well as sorption/desorption processes were suggested as the main factors influencing the dynamic reponse characteristics.
    Additional Material: 10 Ill.
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  • 11
    Electronic Resource
    Electronic Resource
    Berlin : Wiley-Blackwell
    Acta Biotechnologica 20 (2000), S. 31-38 
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: In this paper, the in vivo decolourization of the polymeric dye Poly R-478 by semi-solid-state cultures of Phanerochaete chrysosporium BKM-F-1767 (ATCC 24725) was investigated, employing corncob as a support. In order to stimulate the ligninolytic system of the fungus, the cultures were supplemented with veratryl alcohol (2 mM) or manganese (IV) oxide (1 g/l).Maximum manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) activities of around 2,000 U/l and 400 U/l were attained by the former, whereas the activities reached by the latter were of about 1,500 U/l and 200 U/l, respectively. Furthermore, laccase activity (around 150 U/l) was only detected in manganese (IV) oxide supplemented cultures.The polymeric dye Poly R-478 (0.02 w/v) was added to three-day-old cultures. A percentage of biological decolourization of about 85% was achieved using cultures supplemented with veratryl alcohol, whereas MnO2 cultures showed a rather lower percentage of around 58% after nine days of dye incubation. Moreover, a correlation between MnP activity and Poly R-478 decolourization could be observed, indicating that this enzyme is mainly responsible for dye degradation.In the present work, the in vivo decolourizing capability of the ligninolytic complex secreted by P. chrysosporium was investigated under the above-mentioned cultivation conditions, employing a model compound, such as the polymeric dye Poly R-478.
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  • 12
    Electronic Resource
    Electronic Resource
    Berlin : Wiley-Blackwell
    Acta Biotechnologica 20 (2000), S. 187-187 
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
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  • 13
    Electronic Resource
    Electronic Resource
    Berlin : Wiley-Blackwell
    Acta Biotechnologica 20 (2000), S. 189-201 
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Desulfovibrio vulgaris strain PY1 was isolated from a 3-chlorobenzoic acid (3CBA) degrading anaerobic enrichment culture, using anaerobic Percoll density centrifugation. When grown on pyruvate (20 mM), in the absence of sulphate and under strict anaerobic conditions, this organism converted not only the co-substrates benzoate (BA), 3-amino-BA and 3CBA to the corresponding alcohols but also ten other different halogenated benzoic acids, viz., 4-Cl-, 3-Br-, 4-Br-, 3-I-, 3-F-, 4-F-, 2,4-di-Cl-, 2,5-di-Cl-, 3,4-di-Cl- and 3,5-di-Cl-BA. This was verfied with HPLC and GC/MS spectrometric analyses. The yields of the co-substrate converted after 30 days of growth were between 20% and 88%, depending on the compounds which had been added at initial concentrations of 500 μM. Sulphate, sulphite, thiosulphate and disulphite inhibited the formation of 3-Cl-benzyl alcohol (3CBOH), i.e. a 97 to 99% inhibition, and nitrate and sulphur had no effect (a 7-10% inhibition). In cell-free extracts, the reduction of 3CBA to 3CBOH required strict anaerobic conditions, pyruvate or H2 as electron donors and the addition of methylviologen (MV), FAD, FMN or ferredoxin as electron carriers. The specific activity of the reduction of 3CBA to 3CBOH in crude extract was 5.3 nmol/(mg protein min). The reaction was not inhibited by additions of sulphate or sulphite (5 mM), but was completely inhibited at concentrations of 10 mM 3CBA or 50 mM BA. A carboxylic acid reductase (aldehyde dehydrogenase), which acted on non-activated 3CBA and was responsible for the reduction of 3CBA to 3-Cl-benzaldehyde, was found in the solube fraction (94% of the total activity). These results demonstrate that strain PY1 was able to effectively reduce a wide range of halogenated benzoic acids to the corresponding alcohols.
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  • 14
    Electronic Resource
    Electronic Resource
    Berlin : Wiley-Blackwell
    Acta Biotechnologica 20 (2000), S. 203-218 
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: The recent upsurge in information technology has provided the international community with an easy access to professional journals (e.g. Electronic Journal of Biotechnology at http://www.ejb.org; etc.), discussion groups (e.g. bioenergy@cret.org; digestion@crest.org; etc.) and recently to electronic international conferences (e.g. ICIBS; http://www.cid.harvard.edu/cidbiotech, etc.) as well as a series of biotechnological information material (e.g. http://www.psrast.org, etc.) to stay in contact and receive up-to-date information in biotechnology. There is no doubt that this new technology will be more cost effective in future and reach more people in communities around the globe.This review reports on one such an electronic conference aiming at bridging the communication gap between developed and developing countries. This conference dealt with integrated biosystems and has provided an excellent forum for more than 100 active participants from all regions of the world. As has been demonstrated in this review, the conference was able to show the very different approaches towards the use of biotechnology in developed and developing countries, cold and tropical climate regions owing to their different ecological, economical and societal problems. It also demonstrated very clearly that the field of molecular genetics and/or genetic engineering is not a priority issue in developing countries, but rather the need for clean technologies, multiproduct formation through socio-economic integrated biosystems, e.g. incorporating microbial waste management into agro-industries, in human activities and their roles in creating better health conditions, a better environment and sustain development.It is hoped that this review will lead to a greater use of the electronic facilities available to inform and educate both the northern and the southern communities more readily of their needs and requirements to improve understanding and efforts for a sustainable future.
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  • 15
    Electronic Resource
    Electronic Resource
    Berlin : Wiley-Blackwell
    Acta Biotechnologica 20 (2000), S. 275-288 
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: The purpose and scope of this article is to introduce capable zeolites into downstream processing of natural compounds, especially flavour compounds like 2,5-dimethyl-4-hydroxy-3(2H)-furan-3-one (Furaneol®Furaeol is a registered trademark of FIRMENICH, Ch). The synthesis and the recovery of Furaneol from L-rhamnose are presented. Therefore adsorption isotherms of the zeolites ZSM5 and DAY with varying modules have been determined and adsorption experiments using model and reaction mixtures of Furaneol synthesis were performed and will be discussed.
    Additional Material: 11 Ill.
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  • 16
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: A fermentation process for manufacturing 1,4-piperazinium-(L,L)-dilactate from renewable raw materials and a method for processing this product into L,L-dilactide are described. Lactic acid fermentation with Lactobacillus paracasei was modified in such a way that pH control occurred by using an aqueous solution of piperazine as a correcting agent instead of sodium hydroxide solution. The production of a stoichiometrically composed piperazinium lactate was possible when the pH was 5.0. From 5.0 kg of glucose and 2.15 kg of piperazine, 6.65 kg of 1,4-piperazinium-(L,L)-dilactate were formed in the fermentation process. Separation from fermentation broth, purification and concentration of the product in aqueous solutions were carried out by means of ultrafiltration, nanofiltration and electrodialysis. Total product retention by the membranes used was about 33%. The crystalline salt was obtained by vacuum evaporation. Processing of the 1,4-piperazinium-(L,L)-dilactate into L,L-dilactide was performed in a special glass reactor. A product yield of 70% was achieved. The purified product was characterized by elementary analysis, as well as solubility behaviour, polarity and spectroscopic data. An overall process consisting of the stages fermentation, purification and concentration of piperazinium dilactate as well as cyclization of the latter to dilactide is described.
    Additional Material: 9 Ill.
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  • 17
    Electronic Resource
    Electronic Resource
    Berlin : Wiley-Blackwell
    Acta Biotechnologica 20 (2000), S. 161-168 
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Five different chemical reagents and γ-rays were tested for the sanitization of immobilized biocatalysts with high penicillin G acylase (PGA) activity. The most effective chemical reagents were N-cetyl-N,N,N-trimethylammonium bromide (CTAB) and 2-isopropyl-5-methylphenol (thymol). The optimum concentration of CTAB for the treatment of the immobilized enzyme was 0.25% [w/v] and 1 h, for immobilized cells 0. [w/v] and 3 h. The optimum concentration of thymol for the immobilized enzyme was found to be 0.1% [w/v] and 1 h, for immobilized cells 0.27% [w/v] and 2 h. The optimum dose of γ-rays for the sanitization of the immobilized enzyme was established as 3.2 kGy, for immobilized cells as 4.5 kGy.
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  • 18
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
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  • 19
    Electronic Resource
    Electronic Resource
    Berlin : Wiley-Blackwell
    Acta Biotechnologica 20 (2000), S. 53-64 
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: An advanced primary treatment process for a municipal waste water was systematically studied, using a bio-flocculation-adsorption, sedimentation and stabilzation process (BSS). It was shown that the organic removal efficiency was higher than that of the traditional primary treatment processes but lower than that of the traditional secondary treatment processes. Both adsorption and bio-flocculation played an important role in the removal of pollutants. The activated sludge within the bio-flocculation-adsorption tank could be considered a bio-flocculent which improved the quality of the effluent from the primary treatment process. As the effluent of the BSS process did not meet the requirements for a typical secondary effluent, the process may be regarded as an advanced (or enhanced) primary treatment process, suitable for waste water containing a high concentration of suspended solids and colloidal particles.
    Additional Material: 7 Ill.
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  • 20
    Electronic Resource
    Electronic Resource
    Berlin : Wiley-Blackwell
    Acta Biotechnologica 20 (2000), S. 67-73 
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: The concentration of airborne fungal spores and bacteria as related to room temperature, humidity and occupancy levels within a library building in Singapore was determined. Measurement of indoor air quality with respect to microorganisms is of particular importance in tropical environments due to the extensive use of air-conditioning systems and the potential implications for human health. This study has revealed a number of interesting relationships between the concentrations of fungal spores and bacteria in relation to both environmental and human factors. The levels of fungal spores measured in the indoor environment were approximately fifty times lower than those measured outside, probably because of the lowered humidity caused by air-conditioning in the indoor environment. The variation in fungal spore concentration in the outdoor environment is likely to be due to the diurnal periodicity of spore release and the response to environmental factors such as light temperature and humidity. The indoor concentration of fungal spores in air was not clearly correlated to concentrations measured in air outside of the library building and remained relatively constant, unaffected by the difference in the numbers of occupants in the library. In contrast, the indoor concentrations of bacteria in air were approximately ten times higher than those measured outdoors, indicating a signficant internal source of bacteria. The elevated levels of indoor bacteria were primarily attributed to the number of library occupants. Increased human shedding of skin cells, ejection of microorganisms and particulates from the respiratory tract, and the transport of bacteria on suspended dust particles from floor surfaces probably accounts for the strong positive correlation between occupancy levels and the concentration of bacteria in internal air.
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  • 21
    Electronic Resource
    Electronic Resource
    Berlin : Wiley-Blackwell
    Acta Biotechnologica 20 (2000) 
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
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  • 22
    Electronic Resource
    Electronic Resource
    Berlin : Wiley-Blackwell
    Acta Biotechnologica 20 (2000), S. 96-96 
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
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  • 23
    Electronic Resource
    Electronic Resource
    Berlin : Wiley-Blackwell
    Acta Biotechnologica 20 (2000), S. 87-96 
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Preliminary results on the novel use of the bacterium Thiobacillus ferrooxidans (ATCCJ 3598 and ATCC33020) for the micro-machining (or biomachinig) of metals are reported. Biomachning is a controlled microbiological process to selectively form microstrucutures on a metal work-piece by metal removal (or dissolution) using microorganisms. Applying copper and mild steel as work-pieces, it was shown that the mass removed increased proportionately with machining time. In another experiment, the work-pieces were coated with organic photo-resistive materials to mask (i.e. protect) certain regions of the metlas, thereby defining the microstructure to be formed. The unmasked regions were successfully biomachined; the final machined profile was shown to be similar to the coating image on the original metal. Although biomachining proceeded at a slower rate than chemical machining, the undesired leaching of the metal in the region under the masked area (termed undercutting) was not as severely encountered when compared with the latter. This work demonstrates the potential use of microorganisms for the biomachining of metals. As a “green process”, the innovative use of T. ferrooxidans for the micro-machining of metals opens up the possibility of biomachining as an alternative to conventional metal processing.
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  • 24
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: During the microbial treatment of a sandy model soil artificially contaminated with polycyclic aromatic hydrocarbons (PAHs), a large residual pollution was found. The remainig PAHs were sorbed into the micropores of the soil and were therefore not bioavailable. Using a lab-scale precolator, the microbially pretreated soil was subjected to aftertreatment with surfactants with the aim of further degradation of its pollution. Two commercial nonionic surfatants of the polyethoxylate type, Präwozell F1214/5 N and Sapogenat T-300, were used. The surfactants differ both in their physicochemical properties (CMC value, PAH solubilization capacity, adsorption onto soil) and in their microbial degradability. During aftertreatment under permanently aerobic conditions, only a weak PAH accumulation in the liquid phase was observed, which was due to a low solubilization rate as well as to simultaneous microbial degradation of the dissolved PAHs. Temporary anaerobiosis successfully suppressed the microbial degradation of both the surfactant and the solubilized PAHs, resulting in a more intensive PAH accumulation. But the PAH content of the soil - the essential criterion for evaluating the efficiency of surfactant application - was not decreased to a larger extent with surfactants than without them. To find out why the surfactants failed to act, the surfactant and hydrocarbon distribution among the liquid and solid phases was studied in mixtures of phenantherne-spiked solis and Präwozell-containig liquids; at heavy phenanthrene loading, the aqueous phase was saturated with PAH; at weak loading, it was unsaturated. Model-aided data analysis showed that the soil may contain PAH in two fractions: strongly sorbed into soil pores and, in the case of heavy loading, also weakly attached to the soil surface. The latter is easily extractable, resulting in a PAH-saturated liquid, while strongly adsorbed PAH is only partially dissolved due to competition between the micelles and the soil pores for the PAH. The microbially pretreated soil contains only strongly bound PAHs, which are as difficult to extract by surfactants as they are poorly accessible for microbes.
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  • 25
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Autochthonous bacteriocenoses in semiarid soils in Patagonia were found to be capable of rapidly adapting to high contamination with crude oil. This adaptation at community level is due to the selective enrichment of hydrocarbon-utilizing bacteria always present in these soils. Immediately after a heavy contamination with crude oil, the authochthonous bacteriocenosis contained about 28% hydrocarbon-utilizing bacteria which could be classified into eight ecotypes with characteristic metabolic profiles. Mainly n-alkanes were used as growth substrates of representative strains. After seven months' exposure to crude oil, the bacteriocenosis consisted almost entirely of hydrocarbon-utilizing bacteria. At least fourteen ecotypes were distinguishable, and the majority of representative strains were able to metabolize a broad spectrum of aliphatic and aromatic hydrocarbons. Corresponding to the significant alteration of the physiological diversity, drastic changes to the taxonomic diversity were also found. Whereas at the beginning of the study the autochthonous bacteriocenoses were dominated by GRAM-positive genera of the Actinomycetales (Dietzia, Gordona, Nocardia, Rhodococcus, Streptomyces) with high ecological potency, after just two months' exposure to crude oil, GRAM- negative bacteria (especially Pseudomonas stutzeri) became predominant within the hydrocarbon-utilizing bacteriocenoses accompanied by some GRAM-positive genera of the Actinomycetales with a significantly lower abundance. These findings underline the importance of Pseudomonas and some genera of Actinomycetales for processes of natural attenuation and the technically supported in situ bioremediation of soil polluted by crude oil in Patagonia.
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  • 26
    Electronic Resource
    Electronic Resource
    Berlin : Wiley-Blackwell
    Acta Biotechnologica 20 (2000), S. 149-159 
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Some parameters of the production of an alkaline protease by Rhizopus oryzae in the solid state fermentation of wheat bran were optimized. Using the optimum parameters of an inoculum age of 7 days, an incubation time of 9 days, an amount of CZAPEK-DOX (liquid medium) of 6 ml/g bran and an incubation temperature of 33°C, an activity of 50 U/g bran was achieved. The initial pH of the CZAPEK-DOX medium had little effect. Re-incubation of mouldy bran with only fresh CZAPEK-DOX yielded 3 times total activity compared to single-cycle fermentation. As for the effect of the amount CZAPEK-DOX medium, the water constituent contributed more to activity increase than did the salt component. The ARRHENIUS activation energies were 23 and 7.9 kcal/mole below and above the optimum of 33°C, respectively. In all the studies, along with protease production, variation of protein content and specific activity were also observed. Attempts were made to explain the effects and also gauge their implications for large-scale production.
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  • 27
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
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  • 28
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    Berlin : Wiley-Blackwell
    Acta Biotechnologica 20 (2000) 
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
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  • 29
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Because of the growing utilization of renewable raw materials, the technical use of lignocellulosic fibres from wood and other annual plant materials is becoming increasingly important. The conventional production process of fibreboards is characterized by high-energy consumption and use of ecologically insecure synthetic lesins. Approximately 40 to 45% of the total energy expenditure are used for the thermo-mechanical pulping. Because of high plastication temperatures, an inactive lignin crust on the fibre surface is formed. For that reason, for glueing of the fibres, urea formaldehyde and melamin resins are usually used. The costs for the resin amount to approximately 50% of the entire material costs. In addition, environmental problems are caused. The aim of our investigation is the reduction of energy and resin consumption by enzymatic modification of wood chips and the enzymatic activation of the inherent bonding strength of the material. The first industrial use of fungi for the modification of wood was in the production of “Myco wood”. Pleurothus ostreatus and Trametes versicolor were applied for nonsterile delignification of beech wood. The present investigation of the authors deals with the mycological pre-treatment of wood chips in order to reduce the energy consumption during wood pulping. The screening results favour the brown rotter Gleophyllum trabeum for pinewood (Pinus silvestris) and the white rotter Trametes hirsuta for beech (Fagus silvatica). Both species show resistance against mould fungi. The use of submerged inoculum of these fungi has the advantage over wheat inoculum that the lag phase is less than 12 hours and that the addition of nutrients or fungicides is not necessary. Short-time wood chip incubation results in a 40% decrease of energy consumption during thermo-mechanical pulping and in improved fibreboard properties. Lignin reduction could not be determined by gravimetrical and x-ray microanalysis.Comparative investigations of fibre incubation using laccase, a submerged culture of Trametes versicolor and rape straw fibres show a high increase in bending and tensile strength and an improvement in the hygroscopic properties of glue-free fibre boards for the last two incubation kinds. Similar effects have been obtained incubating pine wood fibres for the production of fibre sheets with enzyme medium of Trichoderma reseei.
    Additional Material: 4 Ill.
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  • 30
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    Berlin : Wiley-Blackwell
    Acta Biotechnologica 20 (2000), S. 335-350 
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: During the 20th century, important relationships developed between the oil industry and both microbiological and biotechnological research. Basic microbiological research has played an important role in both the exploration and production sectors of the oil industry, but as the maturity of the industry has progressed, such contributions have been relegated with respect to their importance. With respect to refining and petrochemicals manufacture, process routes have been extensively researched, but only rarely have the biotechnological solutions developed satisfied the economic criteria that resulted in major investment. In fact, situations exist where investment has occurred, but project life was unrealistically short, suggesting a need for extreme caution when evaluating biotechnological processes for the oil industry. However, as far as engineered processes for both biotreatment and bioremediation are concerned, the fundamental research that has underpinned other areas of hydrocarbon microbiology will finally prove to be of both technical and economic value, in ensuring that the essential needs of treatment, rather than disposal, and restoration, rather than environmental destruction, can be satisfied by the oil and other industries involved in both geochemical manipulation and natural resource exploitation.
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  • 31
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: The influence of different growth-limiting factors - namely the sources of carbon, nitrogen and phosphorus and the dilution (growth) rate - on the ice-nucleation activity of Pseudomonas syringe CCM 4073 was studied. A higher ice-nucleation activity was observed at a lower dilution (growth) rate (D = 0.1 h-1) than at a higher dilution (growth) rate (D = 0.3 h-1). Remarkable differences in ice-nucleation activity were found in its dependence on the growth-limiting factor. The highest ice-nucleation activity was observed under carbon limitation (T90 = -2.7°C), a medium activity under nitrogen limitation (T90 = -5°C) and lowest activity under phosphorus limitation (T90 = -12.3°C). After the addition of excess nitrogen or phosphorus to steady-state cultures, the ice-nucleation activity was restored.
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  • 32
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
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  • 33
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    Berlin : Wiley-Blackwell
    Acta Biotechnologica 20 (2000), S. 65-65 
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
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  • 34
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
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  • 35
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    Berlin : Wiley-Blackwell
    Acta Biotechnologica 20 (2000), S. 41-52 
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Studies were carried out to evaluate the feasibility of using coffee industry residues, viz. coffee husk, coffee leaves and spent coffee ground as substrates in solid state fermentation (SSF) to cultivate edible mushrooms Pleurotus. Eight strains of Pleurotus ostreatus and two strains of Pleurotus sajor-caju were screened on a medium prepared from aqueous extract of coffee husk and agar. Based on best mycelial growth (9.68 mm/day) and biomass production (43.4 mg/plate in 9 days at 24°C), the strain P. ostreatus LPB 09 was selected for detailed studies. SSF was carried out using these substrates under different moisture conditions (45-75%) and spawn rates (2.5-25%). In general, although a 25% spawn rate appeared superior, the 10% spawn rate was recommended for all the three substrates in view of the process economics, as there was not any significant difference in the increase with 10 to 15%. The ideal moisture content for mycelial growth was 60-65% for coffee husk and spent coffee ground, and 60-70% for coffee leaves. The biological efficiency (BE), which is defined as the ratio of the weight of fresh fruiting bodies to the weight of dry substrate, multiplied by 100, and which indicates the fructification ability of the fungus for utilizing the substrate, was best with coffee husk. With coffee husk as the substrate, the first fructification occurred after 20 days of inoculation, and the biological efficiency reached about 97% after 60 days. When coffee leaves were used as the substrate, no fructification was observed even upon prolonged cultivation. With spent ground as the substrate, the first fructification occurred 23 days after inoculation and the biological efficiency reached about 90% in 50 days. There was a significant decrease in the caffeine and tannin contents (61 and 79%, respectively) of coffee husk after 60 days. It was remarkable to observe that caffeine was adsorbed onto the fruiting body (0.157%), indicating that it was not completely degraded by the fungal culture. However, no tannins were found in the fruiting body, indicating that the fungal strain was capable of degrading them. The results showed the feasibility of using coffee husk and spent coffee ground as substrates without any pre-treatment for the cultivation of edible fungi in SSF, and provided one of the first steps towards an economical utilization of these otherwise unutilized or poorly utilized residues.
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  • 36
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    Berlin : Wiley-Blackwell
    Acta Biotechnologica 20 (2000), S. 75-81 
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Serratia marcescens biovar A2/A6 is able to produce a red pigment as a secondary metabolite which has antimicrobial activity. This paper describes its growth and biopigment formation in batch cultures, in media containing different concentrations of lactic acid and beef extract as carbon and nitrogen sources, respectively. An unstructured model has also been developed to describe its growth, lactic acid uptake and biopigment formation. The comparison of simulated and experimental data shows that the proposed model predicts reasonably well the system behaviour over a range of conditions.
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  • 37
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    Berlin : Wiley-Blackwell
    Acta Biotechnologica 20 (2000), S. 97-98 
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
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  • 38
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    Berlin : Wiley-Blackwell
    Acta Biotechnologica 20 (2000) 
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
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  • 39
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    Berlin : Wiley-Blackwell
    Acta Biotechnologica 20 (2000), S. 334-334 
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
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  • 40
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: The impact of hydrocarbon remediation on several enzyme activities (catalase, dehydrogenase, lipase, protease, urease, alkaline phosphomonoesterase, fluorescein diacetate hydrolysis) and microbial properties (biomass-C, respiration, N-mineralization, qCO2, microbial counts) was evaluated in a laboratory study over a period of 10 weeks. A pristine soil was contaminated with diesel oil (10 mg/g soil) or with a mixture of phenanthrene and naphthalene (total amount 1 mg/g soil) and supplemented with inorganic nutrients to give a C:N ratio of 20:1. The corresponding controls consisted of uncontaminated nutrient-supplemented soil. Oil contamination caused a significant initial increase of all biological parameters measured. In the presence of PAHs, biomass-C, respiration, protease activity and heterotrophic counts were significantly enhanced, while urease activity was depressed. N-mineralization was initially, however, reversibly inhibited in the presence of oil and PAHs.The measured parameters behaved differently over time: Biomass-C, respiration and alkaline phosphomonoesterase activity reached a maximum activity after about 2-5 weeks, corresponding to the period during which the majority of hydrocarbons disappeared, and declined thereafter to the background level. Activities of catalase and dehydrogenase also followed this pattern, however, were characterized by fluctuations. Activities of lipase, protease, urease and fluorescein diacetate hydrolysis increased and remained almost constant throughout the incubation period.
    Additional Material: 10 Ill.
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  • 41
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    Berlin : Wiley-Blackwell
    Acta Biotechnologica 20 (2000), S. 351-368 
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: The action resonance theory (ART), a hypothesis based on a logical extension of EINSTEIN's theory of Brownian movement, suggests that the genotype × environment interaction can be modelled as forceful encounters of the gene-products of an organism with its environment. This model has implications for molecular and cell biology, morphogenesis, evolutionary development via mutation, the mechanism of natural selection and overall function of ecosystems, extending SCHRÖDINGER's programme for molecular biology. Action, a thermodynamic property with the same physical dimensions as angular momentum and PLANCK's quantum of action, is proposed to be reversibly generated as a result of the molecular exchange of quanta, which become resonant at equilibrium, corresponding to an optimum degree of entropy and action for living systems. Because the theory can potentially predict solutions to unsolved problems such as the folding of proteins it has strong implications for successful genetic modification of organisms and for biotechnology in general; the design of a programme of research to test this theory is proposed. A key element in this research programme, improving productivity and sustainability, would be the need to select genetically modified strains in the ecological environment or niche in which they are required to function.
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  • 42
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
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  • 43
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Continuous counter-current chromatographic processes have been successfully used in the petrochemical and sugar industry over the last 30 years. Only recently has simulated moving bed (SMB)-technology attracted widespread interest in the pharmaceutical industry, mainly as a very efficient system for chromatographic enantioseparation. The application of this technique to the downstream processing of biotechnological products requires some specific changes to meet the special demands of bioproduct isolation. Production processes are set up on an multi-ton scale, for example, for the purification of fructose with both yield and purity higher than 90%. Examples for other mono- and oligosaccharides are reported. In the purification of fatty acids or fat soluble vitamins, SMB technology under supercritical fluid conditions gives additional benefits and increases the productivity by a factor of four when a pressure gradient is applied. Another field of operation is the isolation of drug compounds from natural sources where different batch- and SMB-chromatographic steps could be successfully combined. First examples are reported for cyclosporine A and paclitaxel isolation. Finally, step-gradient elution modes can be used continuously, as demonstrated for the isolation of monoclonal antibodies.
    Additional Material: 6 Ill.
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  • 44
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 3 (1975), S. 391-400 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Epithelial cells (80-90% “granular” type) were isolated from urinary bladders of Bufo marinus and Rana catesbiana. The inhibitory effect of α-methyl-D-mannoside on fluorescein-labeled concanavalin A (Con A) binding to these cells indicates that they possess specific binding sites for Con A. The lectin also mediates adsorption of erythrocytes to these cells. Both Con A binding and Con A-mediated hem-adsorption to epithelial cells are depressed at 4°C, as compared with cells maintained at 22°C. Elevation of temperature to 37°C, however, enhances hemadsorption independently of alterations in lectin binding. Treatment of cells with antidiuretic hormone (ADH) at 22°C followed by 15 min of incubation at 22° or 37°C before exposure of cells to Con A promotes increments in Con A-mediated hemadsorption, but not in lectin binding, at 22° or 37°C. These hormonal effects are not significant when hemadsorption is assayed at 4°C. Treatment of cells with another octapeptide, angiotensin, elicits a small, but significant, increment in hemadsorption to epithelial cells which is likewise uninfluenced by quantitative changes in lectin binding. Collectively, these data and other independent observations suggest that treatment with octapeptide hormones acts to enhance the redistribution and aggregation of lectin-binding proteins in the membranes of granular epithelial cells from amphibian urinary bladder. Such changes, in turn, may contribute to the alterations in membrane transport properties which characterize the hormonal response.
    Additional Material: 3 Ill.
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  • 45
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 3 (1975), S. 376-390 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: According to current models of muscle contraction (Huxley, H. E., Science 164: 1356-1366 [19691]), motion of flexible myosin crossbridges is essential t o the contractile cycle. Using a spin-label analog of iodoacetamide bound to the subfragment # 1 (S1) region of myosin, we have obtained rotational correlation times (τ2) for this region of the molecule with the ultimate goal of making quantitative measurements of the motion of the crossbridges under conditions comparable to those in living, contracting muscle. We used the newly developed technique of saturation transfer electron paramagnetic resonance spectroscopy (Hyde, J.S., and Thomas, D.D., Ann. N.Y. Acad. Sci. 222:680-692 [1973]), which is uniquely sensitive t o rotational motion in the range of 10-7-10-3 sec. Our results indicate that the spin label is rigidly bound to S1 (τ2 for isolated S1 is 2 × 10-7 sec) and that the motion of the label reflects the motion of the S1 region of myosin. The value of τ2 for the S1 segment of myosin is less than twice that for isolated S1, while the molecular weights differ by a factor of 4, indicating flexibility of myosin in agreement with the conclusions of Mendelson et aL (Biochemistry 12:2250-2255 [1973]). Adding F-actin increases τ2 in either myosin or isolated S1 by a factor of nearly 103, indicating rigid immobilization of S1 by actin. Formation of myosin filaments (at an ionic strength of 0.15 or less) increases τ2 by a factor of 10-30, depending o n the ionic strength, indicating a decrease of the rotational mobility of S1 in these aggregates. The remaining motion is at least a factor of 10 faster than would be expected for the filament itself, suggesting motion of the S1 region independent of the filament backbone but slower than in a single molecule. F-actin has a strong immobilizing effect on labeled S l in myosin filaments (in 0.137 M KC1), but the immobilization is less complete than that observed when F-actin is added t o labeled myosin monomers (in 0.5 M KC1). A spin-label analog of maleimide, attached to the SH-2 thiol groups of S1, is immobilized to a much lesser extent by F-actin than is the label on SH-1 groups. The maleimide label also was attached directly to F-actin and was sufficiently immobilized to suggest rigid binding to actin.
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  • 46
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 3 (1975), S. 112-124 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Evidence for a proposed activation mechanism is summarized. The low rate of ATP cleavage in the resting state of muscle is considered to result from the formation of a stable ring structure involving the two essential sulfhydryl groups on each myosin head and MgATP. Activation is thought to occur by interaction of actin in the vicinity of one of the essential sulfhydryl groups, Thus opening the stable ring leading to rapid dissociation of split products. This idea is consistent with the kinetic scheme of ATP cleavage developed recently by other workers and allows a prediction of the shift in population of intermediate states with changes in solvent conditions. It is also supported by our recent studies on the spatial geometry of the ring. The possibility that other nucleophilic groups may replace the sulfhydryl groups in other contractile systems is considered. The relevance of the ring structure to the tension generating event is discussed on the basis of recent measurements of the rate of contraction of modified (SH1-blocked) actomyosin threads. Results indicate that the ability to form the ring structure is an essential requirement of the contractile process in these systems, and, moreover, that single, modified heads of myosin can act independently to produce the same rate of contraction as native myosin. This latter finding suggests that the myosin duplex exhibits some type of negative cooperativity in the contractile process.
    Additional Material: 7 Ill.
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  • 47
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 3 (1975), S. 146-153 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The extent of actin polymerization has been studied for samples in which the bound nucleotide of the actin was ATP, ADP, or an analog of ATP that was not split (AMPPNP). The equilibrium constants for the addition of a monomer to a polymer end were determined from the concentration of monomer coexisting with the polymer. An analysis of these results concludes that the bound ATP on G-actin provides little energy to promote the polymerization of the actin. AMPPNP was incorporated into F-actin and the interaction of F-actin · AMPPNP with myosin was studied. F-actin · AMPPNP activated the ATPase of myosin to the same extent as did F-actin · ADP. However, the rate of superprecipitation was slower in the case of F-actin · AMPPNP than in the control.
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  • 48
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 3 (1975), S. 256-260 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: 1,N6-Ethenoadenosine diphosphate (∊-ADP) inhibits reverse electron flow (succinate → NAD+ driven by ATP) by competing with ATP, in contrast to ADP which we have shown previously to be a noncompetitive inhibitor. From these and other data it is concluded that the noncompetitive inhibition noted with ADP results from a combination of competitive inhibition plus non- or uncompetitive inhibition, the former occuring at a relatively nonspecific catalytic site and the latter at an extracatalytic site apparently quite specific for ADP.ADP, which stimulates ATP ⇌ H2O and Pi ⇌ H2O exchanges appears to be necessary for inhibition by arsenate of these exchanges. It is suggested that the ATP-supported Pi ⇌ H2O exchange may be predominantly of the medium or intermediate type, depending on the concentrations of the Mg2+ complexes of ADP and Pi. Thus only exchanges involving medium ADP and Pi would be expected to show arsenate sensitivity.
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  • 49
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 3 (1975), S. 261-274 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Highly purified preparations of Streptococcus faecalis ATPase contain a similar but inactive protein detected by prolonged polyacrylamide gel electrophoresis. The inactive protein appears to arise by proteolytic cleavage of the major subunits in the enzyme. By use of a new technique, subunit analysis in SDS gels was performed on the enzyme band and the inactive protein band excised from a polyacrylamide gel after electrophoresis. The results indicated that the ATPase has the composition α3β3γ in which α = 60,000, β = 55,000, and γ = 37,000 daltons. The inactive protein appears to have the composition (f)6 in which f = 49,000 daltons. There is also evidence that the enzyme band contains some slightly modified forms of the ATPase, such as α3β2 (f)γ. The inactive protein lacks the capacity for tight nucleotide binding.Our experiments show that the tight ATPase-nucleotide complex formed in S. faecalis cells (the endogenous complex) behaves differently from the tight complex formed in vitro (the exogenous complex). We prepared a doubly labeled complex containing endogenous 32P-labeled ADP and ATP and exogenous 3H-labeled ADP. We observed that the addition of free nucelotide to the doubly labeled ATPase displaced the exogenous bound ligand from the enzyme but not the endogenous bound nucleotide. We suggest that the displaceable and nondisplaceable forms of the tight ATPase-nucleotide complex correspond to two different conformational states of the enzyme.
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  • 50
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 3 (1975) 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 51
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 3 (1975), S. 1-16 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The choline-containing teichoic acids of pneumococci can be modified by biosynthetic replacement of the choline residues with certain structural analogues, such as ethanolamine (EA) or the N-monomethyl- (MEA) and N-dimethyl- (DEA) amino derivatives of ethanolamine. Cells containing such analogues in their teichoic acids develop pleiomorphic alterations in several physiological properties, which include resistance to detergent-induced lysis and inhibition of cell separation (chain formation). We report here the results of physiological studies on the mechanism of these two phenomena. Our results are summarized in the following: (a) Pneumococci grown on various amino alcohols produce cell walls of identical amino sugar and amino acid composition. (b) Both choline- and EA-containing teichoic acids seem to follow the same conservative pattern of segregation during growth and cell division. (c) Lysis sensitivity of pneumococci requires the juxtaposition of lysissensitive (choline-containing) cell walls and endogenous autolysin at the cell wall growth zone. (d) Upon readdition of choline to ethanolamine-containing cells, lysis sensitivity and catalytically active (C-type) autolysin reappear in the bacteria with the same kinetics. (e) The chains of EA-grown pneumococci contain fully compartmentalized cells and normal cross walls.
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  • 52
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 3 (1975), S. 24-38 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The protein products of at least 21 phage genes are needed for the formation of the tail of bacteriophage T4. Cells infected with amber mutants defective in these genes are blocked in the assembly process. By characterizing the intermediate structures and unassembled proteins accumulating in mutant-infected cells, we have been able to delineate most of the gene-controlled steps in tail assembly. Both the organized structures and unassembled proteins serve as precursors for in vitro tail assembly.We review here studies on the initiation, polymerization, and termination of the tail tube and contractile sheath and the genetic control of these processes. These studies make clear the importance of the baseplate; if baseplate formation is blocked (by mutation) the tube and sheath subunits remain essentially unaggregated, in the form of soluble subunits.Seventeen of the 21 tail genes specify proteins involved in baseplate assembly. The genes map contiguously in two separate clusters, one of nine genes and the other of eight genes. Recent studies show that the hexagonal baseplate is the end-product of two independent subassembly pathways. The proteins of the first gene cluster interact to form a structure which probably represents one-sixth of the outer radius. The products of the other gene cluster interact to form the central part of the baseplate.Most of the phage tail precursor proteins appear to be synthesized in a non-aggregating form; they are converted to a reactive form upon incorporation into preformed substrate complexes, without proteolytic cleavage. Thus reactive sites are limited to growing structures.
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  • 53
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    Journal of Supramolecular Structure 3 (1975), S. 51-60 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Lactoperoxidase-catalyzed iodination selectively labels the two glycoproteins (VP1 and VP2) of Newcastle disease virus. The low-molecular-weight, nonglycosylated major viral protein, VP6, was not iodinated in the intact virus but was iodinated in disrupted virions, suggesting a localization on the inner, rather than the outer envelope surface. Studies on the distribution of virion proteins labeled with 125I and 3H-isoleucine between detergent-soluble and detergent-insoluble fractions show that the virion proteins VP4, VP5, and VP6 are solubilized to a much lesser extent than are VP1 and VP2.
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  • 54
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    Journal of Supramolecular Structure 3 (1975) 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 55
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    Journal of Supramolecular Structure 3 (1975), S. 105-111 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Additional Material: 1 Ill.
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  • 56
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    Journal of Supramolecular Structure 3 (1975), S. 154-161 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: A new approach to the direct estimation of the value of the off constant for dissociation of ATP from myosin subfragment 1 (S1) has been developed. From measurements of the extremely slow rate of release of [32P]-ATP formed from 32Pi by S1 catalysis and the amount of rapidly formed [32P]-ATP tightly bound to S1, the value of the off constant is approximately 2.8 × 10-4 sec-1 at pH 7.4.The concentration dependencies for Pi ⇌ H18 OH exchange and for 32Pi incorporation into myosin-bound ATP give direct measurements of the dissociation constant of Pi from S1. Both approaches show that the enzyme has a very low affinity for Pi, with an apparent Kd of 〉 400 mM.Measurement of the average number of water oxygens incorporated into Pi released from ATP by S1-catalyzed hydrolysis in the presence of Mg2+ suggests that the hydrolytic step reverses an average of at least 5.5 times for each ATP cleaved. With the Ca2+-activated hydrolysis, less than one oxygen from water appears in each Pi released. This finding is indicative of a possible isotope effect in the attack of water on the terminal phosphoryl group of ATP.
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  • 57
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    Journal of Supramolecular Structure 3 (1975), S. 175-180 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Methods are described whereby initial processes of muscular contraction may be investigated in a mammalian preparation, the soleus muscle of the rat. Conditions are chosen so that recovery is avoided. An isometric tetanus is investigated and an energy balance sheet is drawn up. It is found that there is more heat evolved than can be accounted for in terms of measured chemical reaction. This discrepancy is discussed with reference to the similar results that have been obtained using frog muscle.
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  • 58
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    Journal of Supramolecular Structure 3 (1975), S. 169-174 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: From comparative studies of the association with polymeric actin of the bifunctional species heavy meromyosin and its monofunctional constituents, information about the relative freedom of these paired elements can be derived. An isotherm for the former binding process is presented which involves, as an experimentally determinable parameter, the local concentration of a second segment after the first of a pair is attached to the lattice. From combined data for these two association reactions a value of 10-4 M is obtained for this quantity. The large degree of segmental flexibility reported for the free heavy meromyosin is still manifested in the association with actin.
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  • 59
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    Journal of Supramolecular Structure 3 (1975), S. 162-168 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Time-dependent fluorescence depolarization and lifetime studies have been made on myosin subfragment 1 to obtain information about mobility changes and dye environment changes when different nucleotides are added. Data are reported for static and actively hydrolyzing systems containing G- and F-actin. Preliminary data indicate that myosin labeled with the fluorophore 1,5 IAEDANS1-and treated with DTT preserves its actin-activated Vmax. S1 prepared in this manner gives lifetime changes which are nearly identical for all systems studied. S1 labeling without DTT addition gives a pattern of lifetimes similar, though not identical to ESR work. Either type of labeling produces no observable change in the polarization decay, and we set an upper limit of 15% length change for the elongate S1. An unusually long fluorescence decay lifetime for the S1-Mg++ ATP-G-actin system is found which may indicate a new acto-S1 state stabilized by G-actin. The method for obtaining the bound fraction of S1's in the presence of actin is presented and applied to the S1-F-actin-Mg++ ATP system. Qualitative agreement is obtained with other methods.
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  • 60
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    Journal of Supramolecular Structure 3 (1975), S. 17-23 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The temperature-dependent dissociation of neutral salt-soluble collagen into its component chains was measured in 0.6-1.6 M urea solutions at pH 7.3. The temperature-dependent association of the same radiocactively labeled collagen into fibers was measured in 0-0.4 M urea solutions, pH 7.3. The effect of urea on the temperature, Tm(G), for half dissociation into chains was small, and the value extrapolated to zero urea concentration was 39°C. In contrast, the effect of urea on the temperature, Tm(F), for half association into fibers was large, and the value at zero urea concentration was 30°C.We conclude that while body temperature provides excellent conditions for the matching of collagen chains to form molecules, the conditions are not optimal for the formation of highly ordered fibers. The large effects of 0.1 M urea suggest that other factors in vivo may help to destabilize mismatched molecular association during fiber growth. Alternately this might be facilitated by parts of the extension peptides of procollagen.
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  • 61
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    Journal of Supramolecular Structure 3 (1975), S. 39-50 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The porcine neurotubule and its basic subunit were found to be modified in vitro by iodination of amino acids (principally tyrosine) using lactoperoxidase. Iodide ion, H2O2, or lactoperoxidase singly or in any pairwise combination had virtually no effect on neurotubules. However, when all three reagents were present, permitting covalent iodination, it was found that at 0.1 iodotyrosines per tubulin dimer the microtubules unravel to form structures which morphologically resemble strands of protofilaments twisted or wound around each other. These abnormal tubules are stable at room temperature and 4°C. Both monomers of tubulin are labeled to approximately the same extent. Iodinated tubulin (0.1 iodotyrosines/dimer) is unable to assemble in vitro under normal assembly conditions. Heavily iodinated microtubules (8 iodines per tubulin dimer) are similar in morphology to the slightly iodinated structures.
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  • 62
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    Journal of Supramolecular Structure 3 (1975), S. 61-66 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: To reveal the presence of rhodopsin on the surface of the mouse retina, a scanning electron microscope study of the immunolabeling of rhodopsin was attempted. The glutaraldehyde-fixed mouse retina was treated first with rabbit antibodies specific against bovine rhodopsin and then with hemocynin-labeled goat antibodies specific against rabbit antibody. The distribution of hemocyanin label on mouse retina and the technique used for labeling are discussed.
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  • 63
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    Journal of Supramolecular Structure 3 (1975), S. 125-140 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The reactivity of myosin to [14C]-labeled N-ethylmaleimide ([14C] NEM) or to tritium was determined in functionally different frog muscles. The incorporation of [14C] NEM into myosin decreased during isotonic or isometric contractions, as compared to resting muscle. The cysteine residues which were protected during contraction were not involved in the ATPase activity or the actin-binding ability of myosin. Peptide mapping revealed that several residues were protected simultaneously. The incorporation of tritium into the peptide N-H groups of myosin was also decreased during muscle activity. These data support the idea that activation and subsequent contraction of muscle are correlated with structural changes in the myosin molecule.The reactivity of myosin to [14C] NEM was increased when the muscle was stretched to 140% rest length and treated with iodoacetate to deplete ATP. Based on in vitro experiments and on literature data, it is suggested that in the resting muscle myosin contains bound MgATP which decreases the rate of incorporation of [14C] NEM into myosin and that upon the irreversible loss of ATP the rate increases.31P nuclear magnetic resonance signals from a number of phosphates were detected in the intact frog muscle. The data indicated that the minimum concentration of ATP in the muscle is 3 mM, a value which agrees with that of chemical determination. The characteristic chemical shifts, coupling constants, and line widths of ATP in the muscle were considerably altered from that of either free ATP in aqueous solutions or ATP in perchloric acid extracts of muscle.
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  • 64
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    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Recent reports in the literature have indicated a physical association of creatinephosphokinase (CPK) with the tail portion of the myosin molecule. The present paper describes further studies on the interaction of CPK with myosin and myosin fragments, using the techniques of electron paramagnetic resonance (EPR) and nanosecond fluorescence depolarization. From EPR work, spin-labeled CPK appears to interact with myosin, tail-less myosin (heavy meromyosin [HMM]), and myosin heads (subfragment-1 [S1]), the extent of interaction being proportional to the S1 content of myosin or its fragments. Spin-labeled CPK did not evidence interaction with the headless myosin “rods”, with myosin tails (light meromyosin [LMM]), with S2 necks (which connect S1 to the rest of the myosin molecule), or with actin. When a fluorescent dye is directed to the essential ∊-amino group of CPK, nanosecond fluorescence depolarization studies indicate a substantial interaction with myosin, HMM, and S1, but very little with F-actin. When the “fast-reacting” thiol of the S1 moiety or the “essential thiol” of CPK was labeled with either a fluorescent dye or a spin label, no interaction between CPK and myosin (or S1) was detected.
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  • 65
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    Journal of Supramolecular Structure 3 (1975) 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 66
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    Journal of Supramolecular Structure 3 (1975), S. 510-519 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The effect of substrate on the far UV (185-250 nm) and near UV (250-325 nm) circular dichroism (CD) of myosin and heavy meromyosin (HMM) was studied. The following results were obtained with the addition of ATP (during various conditions of hydrolysis), ADP, and pyrophosphate: (1) no changes were observed in the far UV CD, (2) ATP and ADP perturbed the near UV CD only at spectral regions below 280 nm coinciding with the regions of their optical activity, (3) the optically inactive pyrophosphate caused no change in the near UV CD, and (4) myosin and HMM gave exactly the same results. These results suggest that myosin-substrate interaction in vitro does not result in a delocalized conformational change.
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  • 67
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    Journal of Supramolecular Structure 3 (1975), S. 80-89 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Fluorescent polymers were obtained by oxidizing partly emulsified linolenic acid with different oxidants. The speed of formation of polymers differed for the various oxidants, and the difference was not a simple function of the oxidation potential. The speed of polymerization also depended on the nature of the emulsion.The presence of egg albumen in the emulsion enhanced polymer formation with all oxidants. When the oxidants used are arranged in the order of decreasing speed of polymer formation, the order is different in the presence of albumen from what it is in the absence of albumen.With different oxidation catalysts most antioxidants and amino acids tested enhanced polymerization. In oxidation with ferric ions, with K-dichromate, and without added oxidants the only antioxidants which delayed polymerization were “inhibitors”. “Retarders” enhanced polymerization. With KMnO4 slight delay was caused by some retarders.The findings indicate that not only oxidation catalysts, but also proteins, amino acids, and antioxidants enhance polymerization. The possibility is suggested that in animal cells lipid pigment formation might represent a mechanism for neutralizing free radicals.
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  • 68
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    Journal of Supramolecular Structure 3 (1975), S. 304-313 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: EPR and water proton relaxation rate (1/T1) studies of partially (40%) and “fully” (90%) purified preparations of membrane-bound (Na++K+) activated ATPase from sheep kidney indicate one tight binding site for Mn2+ per enzyme dimer, with a dissociation constant (KD = 0.88 μM) in agreement with the kinetically determined activator constant, identifying this Mn2+-binding site as the active site of the ATPase. Competition studies indicate that Mg2+ binds at this site with a dissociation constant of 1 mM in agreement with its activator constant.Inorganic phosphate and methylphosphonate bind to the enzyme-Mn2+ complex with similar high affinities and decrease l/T1 of water protons due t o a decrease from four to three in the number of rapidly exchanging water protons in the coordination sphere of enzyme-bound Mn2+. The relative effectiveness of Na+ and K+ in facilitating ternary complex formation with HPO2-4 and CH3PO2-3 as a function of pH indicates that Na+ induces the phosphate monoanion t o interact with enzyme-bound Mn2+, while K+ causes the phosphate dianion to interact with the enzyme-bound Mn2+. Thus protonation of an enzyme-bound phosphoryl group would convert a K+-binding site to a Na+-binding site. Dissociation constants for K+ and Na+, estimated from NMR titrations, agreed with kinetically determined activator constants of these ions consistent with binding t o the active site.Parallel 32Pi-binding studies show negligible formation (〈 7%) of a covalent E-P complex under these conditions, indicating that the NMR method has detected an additional noncovalent intermediate in ion transport. Ouabain, which increases the extent of phosphorylation of the enzyme to 24% at pH 7.5 and t o 106% at pH 6.1, produced further decreases in l/T 1 of water protons. Preliminary 31P-relaxation studies of CH3PO2-3 in the presence of ATPase and Mn2+ yield an Mn to P distance (6.9 ± 0.5 Å) suggesting a second sphere enzyme-Mn-ligand-CH3PO2-3 complex.Previous kinetic studies have shown that T1+ substitutes for K+ in the activation of the enzyme but competes with Na+ at higher levels. From the paramagnetic effect of Mn2+ at the active site on the enzyme on I/T1 of 205T1 bound at the Na+ site, a Mn2+ to T1+ distance of 4.0 ± 0.1 Å is calculated, suggesting the sharing of a common ligand atom by Mn2+ and T1+ on the ATPase. Addition of P. increases this distance to 5.4 Å consistent with the insertion of P between Mn2+ and T1+. These results are consistent with a mechanism for the \documentclass{article}\pagestyle{empty}\begin{document}$ (\mathop {\rm N}\limits^{\rm i} {\rm a}^{\rm + } {\rm + K}^ +) $\end{document}-ATPase and for ion transport in which the ionization state of Pi at a single enzyme active site controls the binding and transport of Na+ and K+, and indicate that the transport site for monovalent cations is very near the catalytic site of the ATTase. Our mechanism also accounts for the order of magnitude weaker binding of Na+ compared to K+.
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  • 69
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    Journal of Supramolecular Structure 3 (1975), S. 103-103 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 70
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    Journal of Supramolecular Structure 3 (1975), S. 521-521 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 71
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    Journal of Supramolecular Structure 3 (1975), S. 248-255 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have partially purified active delta and epsilon subunits of the E. coli membranebound Mg2+ -ATPase (ECF1). Treating purified ECF1 with 50% pyridine precipitates the major subunits (α, β, and γ) of the enzyme, but the two minor subunits (δ and ∊), which are present in relatively small amounts, remain in solution. The delta and epsilon subunits were then resolved from one another by anion exchange chromatography. The partially purified epsilon strongly inhibits the hydrolytic activity of ECF1. The epsilon fraction inhibits both the highly purified five-subunit ATPase and the enzyme deficient in the δ subunit. The latter result indicates that the delta subunit is not required for inhibition by epsilon. By contrast, two-subunit enzyme, consisting chiefly of the α and β subunits, was insensitive to the ATPase inhibitor, suggesting that the γ subunit may be required for inhibition by epsilon.The partially purified delta subunit restored the capacity of ATPase deficient in delta to recombine with ATPase-depleted membranes and to reconstitute ATP-dependent transhydrogenase. Previously we reported (Biochem. Biophys. Res. Commun. 62:764 [1975]) that a fraction containing both the delta and epsilon subunits of ECF1 restored the capacity of ATPase missing delta to recombine with depleted membranes and to function as a coupling factor in oxidative phosphorylation and for the energized transhydrogenase. These reconstitution experiments using isolated subunits provide rather substantial evidence that the delta subunit is essential for attaching the ATPase to the membrane and that the epsilon subunit has a regulatory function as an inhibitor of the ATPase activity of ECF1.
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  • 72
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    Journal of Supramolecular Structure 3 (1975), S. 333-337 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Myosin catalyzed exchange between 32Pi and ATP in reaction medium during its enzymatic hydrolysis of ATP only by a very small amount. Addition of actin increased to a great extent the rate of incorporation of 32Pi in the presence of Mg. Glycerinated smooth muscle fibers also exhibited the ability to exchange 32Pi and ATP upon the application of external force (repeated stretching and releasing). A schematic mechanism of the action of actin and external force on acceleration of 32Pi incorporation is proposed and the importance of the M*-ADP complex for force generation is suggested.
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  • 73
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    Journal of Supramolecular Structure 3 (1975), S. 315-322 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: From transient kinetic studies of the Mg2+-dependent adenosine triphosphatase of myosin subfragment 1, prepared from rabbit skeletal muscle, a seven-step mechanism has been proposed. Features of this mechanism include two-step processes for ATP and ADP binding in which the binary complex isomerizes in addition to a rapid nucleotide association step. In the case of ATP a large negative standard free energy change is associated with the isomerization. An overall rate-limiting isomerization of the myosin-product complex prior to product release has been identified. Studies on the mechanism of cleavage of ATP bound to the active site indicate the process is readily reversible and can account for the observation that more than one oxygen of the product phosphate arises from water. This proposal has been substantiated by the finding that the oxygen atoms of the γ-phosphoryl group of bound ATP also undergo extensive exchange with water.
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  • 74
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    Journal of Supramolecular Structure 3 (1975), S. 361-367 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The binding of ADP to heavy meromyosin has been studied by microcalorimetry. Minute amounts of myokinase interfere with binding measurements, but by selection of appropriate conditions, we can estimate that the value of the apparent ΔHbinding lies between -1.0 and -3.0 kcal per mole of ADP bound (0.3 M KCl, 2 mM MgCl2, 20 mM Tris, pH 8.00, 20°C). Values of ΔHbinding reported to date are an order of magnitude larger, and we suggest that these values are artifactual results due to myokinase contamination.
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  • 75
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    Journal of Supramolecular Structure 3 (1975), S. 348-353 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Structural changes in myosin may take place within the heavy chains, or may represent rearrangement of subunits. Changes induced by ATP and possibly restricted to the heavy chains are indicated by several different optical studies, including changes in absorbence of aromatic residues (1), changes in the fluorescene of dyes coupled to myosin (2), changes in electron spin resonance spectrum (3), and changes in the intrinsic fluorescence of tryptophan residues (4, 5). A calcium dependent subunit rearrangement in scallop myosin is indicated by fluorescene depolarization studies (Cheung and Szent-Györgyi, unpublished observations). In scallop muscles, calcium regulates contraction by directly reacting with myosin (6), and the changes in the quaternary structure of myosin are likely to be related in the way calcium control works.At first I will disucss the changes of fluorenscene that Drs. Weber, Fasman, and I have described several years ago (4). Then I will speculate on the implications of a subunit rearrangement on the mechanism of regulatory light chain funciton.
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  • 76
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    Journal of Supramolecular Structure 3 (1975), S. 354-360 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The interaction of myosin and paramyosin was investigated by enzymological and ultrastructural techniques. The actin-activated Mg+2 ATPase of rabbit skeletal muscle myosin can be inhibited by clam adductor paramyosin. Both proteins must be rapidly coprecipitated to form filaments for this inhibition. Slowly formed cofilaments are fully activatable by F-actin. In both cases, the cofilaments possess unique structural characteristics when compared to homofilaments.The mode of inhibition appears to be competitive when different concentrations of paramyosin and F-actin are compared. The apparent affinity of the myosin heads for actin is reduced by the presence of paramyosin within rapidly reconstituted thick filaments. These results suggest that paramyosin may serve as part of a relaxing mechanism within invertebrate muscles. It is unlikely that paramyosin plays a role in the initiation and maintenance of catch within specialized molluscan muscles.
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  • 77
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    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 78
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    Journal of Supramolecular Structure 3 (1975), S. 181-191 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The changes in birefringence in the rigor to relax transition of single Triton-extracted rabbit psoas muscle fibers have been investigated. The total birefringence of rigor muscle fibers was dependent on sarcomere length and ranged from (1.46 ± 0.08) × 10-3 to (1.60 ± 0.06) ± 10-3 at sarcomere lengths from 2.70 μm to 3.40 μm. An increase in total birefringence was measured dependent on sarcomere length when 55 single fibers were relaxed from the rigor state with Mg-ATP. Pyrophosphate relaxation produced a smaller increase in retardation when compared to Mg-ATP. The expected change in intrinsic birefringence during the rigor to relax transition was calculated assuming a hinge function of the subfragment 2 moiety of myosin. The changes in birefringence during isometric contraction and relaxation have been discussed in relation to possible structural changes.
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  • 79
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    Journal of Supramolecular Structure 3 (1975), S. 201-213 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Energy conservation and uncoupling in mitochondria are examined in the light of three important new findings: (a) Studies with the photoaffinity-labeling uncoupler 2-azido-4-nitrophenol have shown that mitochondria contain a specific uncoupler binding site (apparently a polypeptide of Mr = 30,000 ± 10%). (b) This site fractionates into an enzyme complex (complex V), which is capable of oligomycin- and uncoupler-sensitive ATP-Pi exchange. It is absent from electron transfer complexes I, III, and IV, which represent segments of the respiratory chain containing coupling sites 1, 2, and 3, respectively. (c) Trinitrophenol is a membrane-impermeable uncoupler (uncouples submitochondrial particles, but not mitochondria) and a poor protonophore. There is an excellent correlation between the uncoupling potencies and the affinities of uncouplers for the mitochondrial uncoupler-binding site. There is no correlation between uncoupling potency and protonophoric activity of uncouplers when a membrane-permeable uncoupler is compared with a membrane-impermeable one.
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  • 80
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    Journal of Supramolecular Structure 3 (1975), S. 192-199 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The structural flexibility of creatine kinase has been investigated with the covalent hydrophobic probe 2-[4′-(2″-iodoacetamido) phenyl] aminonaphthalene-6-sulfonic acid (IAANS) which reacts at vastly different rates with the two subunits to give a protein conjugate with fluorescence characteristic of reaction with a site in a hydrophobic cleft. Binding of purine nucleotides greatly enhances the probe fluorescence while pyrimidine nucleotides quench the fluorescence. Small anions bind to nucleotide-free creatine kinase near the location of the transferable phosphoryl group and quench both the IAANS fluorescence of modified creatine kinase and the tryptophan fluorescence of native creatine kinase. Chloride and nitrate non-competitively inhibit MgADP binding both with and without creatine. Fluorescence energy transfer demonstrates that the active sites of creatine kinase are well separated and become further apart after the nucleotide-induced conformational change.
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  • 81
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    Journal of Supramolecular Structure 3 (1975), S. 214-221 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Evidence is presented that mitochondrial ATPase has two types of sites that bind adenine nucleotides. The catalytic site, C, binds the substrates ATP, GTP, or ITP and the inhibitor guanylyl imidodiphosphate (GMP-PNP). A second type of site, R, binds ATP, ADP, adenylyl imidodiphosphate (AMP-PNP), and the chromium complexes of ATP or ADP. All of these substances binding to the R site inhibit the hydrolysis of ATP in a competitive manner; their inhibition of hydrolysis of ITP and GTP is noncompetitive. GMP-PNP inhibits oxidative phosphorylation in submitochondrial particles but AMP-PNP does not. The localization on mitochondrial membranes of sites for the binding of various antibiotics that inhibit oxidative phosphorylation is discussed.
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  • 82
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    Journal of Supramolecular Structure 3 (1975), S. 231-241 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The membrane bound coupling factor-latent ATPase was solubilized from the membrane vesicles of Mycobacterium phlei by using 0.25 M sucrose or low ionic strength buffer. Purification of the solubilized enzyme by use of Sepharose-ADP conjugate gel yielded a homogenous preparation of latent ATPase which was purified about 216-fold in a single step with an 84% yield. The enzyme exhibits a specific activity of 39 μmoles of ATP hydrolyzed per min per mg protein. The purified enzyme exhibits coupling factor activity. Electrophoresis in two dissociating solvent systems indicates that the enzyme contains at least three major polypeptides of molecular weights 56,000, 51,000 and 46,000 daltons, and two minor polypeptides of 30,000 and 17,000 daltons. Equilibrium binding studies of ADP with purified coupling factor-latent ATPase reveal the presence of two nucleotide binding sites per molecule with an apparent Ka of 8.1 × 10-5 M.By use of affinity chromatography, another latent ATPase has been isolated from the solubilized enzyme, which does not exhibit coupling factor activity.
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  • 83
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    Journal of Supramolecular Structure 3 (1975), S. 284-296 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The tightly bound nucleotides of the beef-heart mitochondrial ATPase are released during cold inactivation followed by ammonium sulfate precipitation. During incubation at 0°C the sedimentation coefficient (s20 W) of the ATPase first declines from 12.1 S to 9 S. Prolonged incubation or precipitation with ammonium sulfate leads to dissociation of the 9 S component into subunits with s20 W of 3.5 S. The 9 S component still bears bound nucleotides which exchange more extensively and rapidly with added nucleotides than those bound to the active 12.1 S component. The bound nucleotides are lost when the 9 S form dissociates into the smaller subunits. Thus, firm binding of nucleotides is a property of the quarternary structure of the enzyme.The exchangeability of the nucleotides bound to the ATPase of chloroplast membranes is greatly increased in membranes illuminated in the presence of pyocyanine. Pi can exchange into both the β and γ positions of the bound nucleotides when the membranes are energized in the presence of Mg2+. The exchange of the nucleotides and the incorporation of Pi are insensitive to the inhibitor Dio-9 but are inhibited by the uncoupler S13.Abbreviation: S13, 5-chloro-3-t-butyl-2′-chloro-4′nitrosalicylanilide. This inhibition by S13 parallels that of the inhibition of photosynthetic phosphorylation. These findings are discussed with regard to our hypothesis that electron transfer causes release of preformed tightly bound ATP from the ATPase by inducing a conformational change.
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  • 84
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    Journal of Supramolecular Structure 3 (1975), S. 323-332 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The metal ion requirement of myosin-ADP binding was investigated by use of Mn2+. Mn2+ binds to two sets of noninteracting sites on myosin which are characterized by affinity constants of 106 and 103, M-1 at 0.016 M KCl concentration. The maximum number of sites is 2 for the high affinity and 20-25 for the low affinity set. Binding of Mn2+ to the high affinity sites increases the affinity of ADP binding to myosin.F-actin inhibits ADP binding (Kiely, B., and Martonosi, A., Biochim. Biophys. Acta 172: 158-170 [1969]), but even at F-actin concentrations much higher than that required to saturate the actin binding sites of myosin or its proteolytic fragments, significant ADP binding remained. The actin insensitive portion of ADP binding was inhibited by 10-4 M inorganic pyrophosphate or ATP. The results are discussed on the basis of a model in which actin and ADP bind to myosin at distinct but interacting sites.
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  • 85
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    Journal of Supramolecular Structure 3 (1975), S. 67-79 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Free ribosomes extracted from hamster cells and 28S RNA purified from these ribosomes are known to form dimers. We find that spleen phosphodiesterase inhibits ribosomal dimer formation, but only when a free 5′-hydroxyl end group, produced by the action of alkaline phosphatase, is present. Hence, formation of dimer ribosomes probably involves interaction at or near the phosphorylated 5′-ends of 28S RNA.Dimer RNA molecules show a modal length, when measured on electron micrographs, of 2.1μm, which is about double the length of 28S RNA. Electron micrographs of 115S dimer ribosomes often show profiles consistent with our interpretation that in dimers the 28S RNA chains are loosely linked by their 5′-ends.
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  • 86
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    Journal of Supramolecular Structure 3 (1975), S. 338-347 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The results of energy transfer experiments on the proximity of six sites on the globular head region of myosin are discussed. A large hydrophobic crevice has been detected on each myosin head which is sufficiently large to accommodate six aromatic rings simultaneously. In the crevice is located a thiol residue not involved in activation of myosin Ca2+ ATPase and a lysine residue which is specifically trinitrophenylated with 2, 4, 6-trinitrobenzenesulfonic acid. A second sulfhydryl whose modification activates the Ca2+ ATPase is located near the hydrophobic thiol site. The tryptophan whose fluorescence is enhanced by ATP binding is sufficiently close to the thiols and lysine residue to quantitatively transfer its energy to probes at these sites. The site of myosin ATPase has been tentatively located as being near the other five sites by energy transfer to or from synthetic chromophoric substrates. Implications of these results on the possibility of determining the location of the myosin light chain and actin binding sites are discussed.
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  • 87
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    Journal of Supramolecular Structure 3 (1975) 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 88
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    Journal of Supramolecular Structure 3 (1975), S. 368-375 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In 2 mM MgATP, 0.08 ionic strength and 1 mM free Mg++ cardiac myofibrils bound 3.5 nmoles Ca/mg protein at maximal ATPase activation. Significant amounts of Ca were also bound to cardiac myosin with these same conditions. By subtraction of this myosin-bound Ca we obtained an estimate of 4 moles Ca bound per mole of myofibrillar troponin at maximal ATPase. We found, however, that Ca activation of myofibrillar ATPase could be estimated assuming that only two of troponin's Ca-binding sites are engaged in regulation of crossbridge activity. Increase in MgATP from 0.3 to 5.0 mM raised the free Ca, giving half-maximal isometric tension or ATPase. Although part of this shift is most probably due to changes in the number of rigor (nucleotidefree) actin-myosin linkages, the rightward shift of the free Ca++-activation relation with increase in MgATP from 2 to 5 mM appears to be due to effects of active (nucleotide-containing) actin-myosin linkages.
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  • 89
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    Journal of Supramolecular Structure 3 (1975), S. 401-414 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The ultrastructural deformation technique has been used to study various membranes, including several claimed to show subunit structure. With localized deformation fibers ca. 100-300 Å in diameter and up to 4,000 Å long were found extending across the cracks parallel to the draw direction in all membranes. Lipid extraction and proteolytic enzyme (papain) treatment of membranes has shown that the fibers are protein in nature. Deformation of membranes while still we showed no significant change in the appearance of the fibers compared with those obtained from deformation of membranes after air drying, indicating that formation of fibers is not an artifact due to dehydration. There must be extensive interaction between the protein molecules and thus we indicate that the results do not agree with current suggestions that membranes are composed of individual protein subunits immersed in a lipid bilayer.
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  • 90
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    Journal of Supramolecular Structure 3 (1975), S. 415-425 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Using a rapid method of preparation, spectrin has been isolated from human erythrocytes and its ATPase activity investigated. The ATPase activity with calcium has two distinct components, -one with optimal activity when calcium and ATP are of equal concentration (low-Ca-ATPase) and another which is activated above 1 mM CaCl2 and is maximal at 100 mM CaCl2. There is also a Mg-ATPase with maximal activity at 10 mM MgCl2. The high-Ca-ATPase of spectrin, but not the low-Ca-ATPase, is inhibited by magnesium, while the Mg-ATPase is inhibited by Ca in excess of ATP. None of these activities exhibits the calcium-stimulated magnesium-dependent activity characteristic of the red cell calcium pump.
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  • 91
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    Journal of Supramolecular Structure 3 (1975), S. 448-468 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The synthesis of ATP analogs containing a photoactive aryl azido grouping coupled to the 3′ hydroxyl of ATP is described. The potential effectiveness of these analogs in the investigation of nucleotide-binding regions is outlined and this effectiveness demonstrated by their photodependent inhibition of subfragment 1 ATPase. The use of 14C-labeled azido ATP demonstrates an almost stoichiometric covalent binding of the analog.Because of their potential application to other systems, a number of reactions describing the reactivity of the 3′ hydroxyl of the nucleotide ribose are outlined in an Appendix.
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  • 92
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    Journal of Supramolecular Structure 3 (1975), S. 469-478 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Tightly bound adenine nucleotides are removed from multiple binding sites on beef heart mitochondrial ATPase (F1) by chromatography on columns of Sephadex equilibrated with 50% glycerol. Release of nucleotides from the enzyme is associated with large decreases in sedimentation velocity (from 11.9 S to 8.4 S) which may be observed in concentrated solutions of polyols. Polyol-induced conformational changes are reversed when the enzyme is returned to dilute buffers. The nucleotide-depleted enzyme restores oxidative phosphorylation in F1 -deficient submitochondrial particles. Reconstitution of nucleotide-depleted F1 with the ATP analog (adenylyl-imidodiphosphate) (AMP-PNP), almost 5 moles of AMP-PNP per mole of enzyme, results in preparations with substantially inhibited ATPase activity which nevertheless restores oxidative phosphorylation and the 32Pi-ATP exchange reaction in F1 -deficient submitochondrial particles. Incubation of the analog-labeled enzyme with ATP and Mg++ results in partial displacement of the analog and a time-dependent recovery of ATPase activity.
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  • 93
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    Journal of Supramolecular Structure 3 (1975), S. 90-101 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Bovine cerebral white matter homogenates were oxidized by various oxidizing solutions of equal molarity and subsequently extracted with water. Most of the oxidants tested (K-dichromate, FeCl3, H2O2, O2, and chloroperbenzoic, ascorbic, performic, and periodic acids) rendered the various myelin constituents less extractable than the constituents of unoxidized control homogenates. KMnO4, and to a lesser degree hemoglobin, rendered myelin constituents more extractable with water than those of the control homogenates. The findings indicate that most of the oxidants produced stabilization of the lamellar pattern, probably through cross-linking and polymerization. KMnO4 and hemoglobin caused labilization and breakdown of the membranous structure. Proof that stabilization of membranes is caused by some oxidants and that fragmentation occurs by the action of KMnO4 and hemoglobin was obtained by the light-scattering technique and by electron microscopy of the oxidized homogenates. Evidence obtained indicated that formation of hydrophobic end groups during oxidation favors polymerization, while prevalence of hydrophilic groups is associated with fragmentation.
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  • 94
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    Journal of Supramolecular Structure 3 (1975), S. 275-283 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Rat liver mitochondria were treated with ethylacetimidateAbbreviations: SDS, sodium dodecylsulfate; DMS, dimethylsuberimidate; EA, ethylacetimidate; MBI, methylbutyrimidate; TMPD, N, N′, N′-tetramethyl-p-phenylenediamine. and methylbutyrimidate, monofunctional imidates, and with dimethylsuberimidate, a bifunctional imidate, and the effects on structure and function studied. Mitochondria treated with 5 mM dimethylsuberimidate or greater did not respond osmotically when placed in deionized water. Sodium dodecylsulfate-polyacrylamide gel electrophoresis revealed that at concentrations 〉 5 mM dimethylsuberimidate nearly all mitochondrial polypeptides failed to enter 6% gels, indicating crosslinking of both membrane and soluble proteins. Extensive amidination by ethylacetimidate and methylbutyrimidate had little effect on ascorbate-tetramethylphenylenediamine oxidase while extensive inhibition resulted from dimethylsuberimidate treatment. The possible involvement of molecular motion in electron transport is discussed.
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  • 95
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    Journal of Supramolecular Structure 3 (1975), S. 222-230 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Mitochondrial ATPase from rat liver mitochondria contains multiple nucleotide binding sites. At low concentrations ADP binds with high affinity (1 mole/mole ATPase, KD = 1-2 μM). At high concentrations, ADP inhibits ATP hydrolysis presumably by competing with ATP for the active site (KI = 240-300 μM). As isolated, mitochondrial ATPase contains between 0.6 and 2.5 moles ATP/mole ATPase. This “tightly bound” ATP can be removed by repeated precipitations with ammonium sulfate without altering hydrolytic activity of the enzyme. However, the ATP-depleted enzyme must be redissolved in high concentrations of phosphate to retain activity. AMP-PNP (adenylyl imidodiphosphate) replaces tightly bound ATP removed from the enzyme and inhibits ATP hydrolysis. AMP-PNP has little effect on high affinity binding of ADP. Kinetic studies of ATP hydrolysis reveal hyperbolic velocity vs. ATP plots, provided assays are done in bicarbonate buffer or buffers containing high concentrations of phosphate. Taken together, these studies indicate that sites on the enzyme not directly associated with ATP hydrolysis bind ATP or ADP, and that in the absence of bound nucleotide, Pi can maintain the active form of the enzyme.
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  • 96
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    Journal of Supramolecular Structure 3 (1975), S. 242-247 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: A purified preparation of the oligomycin-sensitive ATPase from yeast mitochondria has been shown to elicit an oligomycin- and uncoupler-sensitive ATP-32Pi exchange in the presence of phospholipids. Reconstitution was normally achieved by dialysis of an ATPase-phospholipid-cholate mixture. Following this procedure, vesicles with diameters between 200 and 1,500 Å were seen by electron microscopy. As in mitochondria, ATPase activity in the reconstituted system was stimulated by a range of uncouplers which inhibited ATP-32Pi exchange. These and other findings suggest that the coupling mechanism may still be intact within the ATPase complex.
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  • 97
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    Journal of Supramolecular Structure 3 (1975), S. 297-303 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Crosslinking of membrane proteins of Escherichia coli with dithiobis (succinimidyl propionate) (DSP) resulted in loss of several enzyme activities including the Ca2+, Mg2+-activated ATPase. This enzyme was crosslinked by DSP to the membrane and was not released by dialysis at low ionic strength in the absence of dithiothreitol which could cleave the crosslinking group. DSP inactivated both phosphohydrolase and coupling activities of the solubilized ATPase. Loss of hydrolytic activity could be correlated with the extent of reaction of the α and/or β subunits of the enzyme. The loss of coupling activity appeared to be associated with modification of the γ and/or δ subunits.
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  • 98
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Fourteen proteins from the large subunit of Escherichia coli ribosomes were analyzed in an improved sequenator. In addition to our previously described modifications of a Beckman sequenator, new valves which work free of a dead volume were constructed. By this and the previous improvements (e.g., a new vacuum system with a recorder, cool traps, automatic conversion) much better results were obtained than before. It was even possible to use (in addition to the standard methods, e.g., thin-layer chromatography and amino acid analysis) mass spectrometry without preceding gas chromatography for identification of the released PTH amino acids. Our experience with the various methods, especially mass spectrometry, is described and the techniques are compared. The results obtained by the described methods on the amino acid sequences of the 14 ribosomal proteins are summarized.
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  • 99
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    Journal of Supramolecular Structure 3 (1975), S. 479-497 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The sodium and potassium ion pump is an intrinsic enzyme of plasma membranes. In these experiments it was driven backward in a transient two-step operation involving, first, phosphorylation of the enzyme from inorganic phosphate, and second, transfer of the phosphate group from the enzyme to ADP upon addition of a high concentration of Na+. There was no evidence of a significant concentration gradient across the membranes. Na+ presumably reached the solutions on both faces of the membrane simultaneously and provided the energy for synthesis simply as a consequence of ligand binding. An interaction free energy between the free energy of the binding of Na+ presumably reached the solutions on both faces of the membrane simultaneously and provided the energy for synthesis simply as a consequence of ligand binding. An interaction free energy between the free energy of the binding of Na+ and the free energy of hydrolysis of the phosphate group on the enzyme was estimated. The experiments also suggested a feature of the transport mechanism. This is control by phosphorylation of access pathways from the solutions in contact with the faces of the membrane to an active center for cation binding. In the dephosphoenzyme access would be to the intracellular solution and in the phosphoenzyme access would be to the extracellular solution.
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  • 100
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    Journal of Supramolecular Structure 3 (1975), S. 498-509 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Chromatophores of Rhodospirillum rubrum and spinach chloroplasts contain firmly bound ATP that is rapidly labeled along with ADP in the presence of 32Pi and endogenous nucleotides. The labeling is not entirely dependent on light. In chloroplasts three types of bound ATP can be defined methodologically by their extraction properties: buffer-soluble; acid-soluble; and SDS-soluble or firmly bound ATP. Extensive washing of the chloroplasts does reduce buffer-soluble but not acidsoluble and firmly bound APT. Buffer-soluble [32P] ATP is almost exclusively γ labeled while acid-soluble and firmly bound ATP are labeled in the β and γ position equally. CCCP, desaspidin, and phlorizin do not inhibit the labeling of firmly bound ATP, whereas the phosphorylation is almost abolished. However, EDTA and NEM pretreatments of the choloroplasts affect both reactions similarly.The postillumination [32P] ATP synthesis with chromatophores can be inhibited by adding ATP to the incubation mixture after illumination if 32Pi is included only during the dark incubation, but is without effect if 32Pi is present only during illumination. On the other hand, ADP added after illumination inhibits post-illumination [32P] ATP formation in both chromatophores and chloroplasts only if 32Pi is present during illumination. The data can be explained by a coupling factor having two sites, as proposed previously on the basis that firmly bound ATP does not transfer its phosphoryl group but seems to drive a synthesis of acid-soluble ATP which incorporates free phosphate (Lutz, H. U., Dahl, J. S., and Bachofen, R.), Biochim. Biophys. Acta 347:359 [1974].
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