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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Planta 196 (1995), S. 1-6 
    ISSN: 1432-2048
    Keywords: Acid-base loading ; Nitrate reductase ; pH regulation (intracellular) ; Protein phosphorylation ; Spinacia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The effect of acid or base-loading of spinach (Spinacia oleracea L.) leaf discs on the activation status of nitrate reductase (NR) in the dark and in the light was investigated. Activity of NR (NRA), measured in crude extracts of leaf discs with removed lower epidermis, which had been floating on Mes-buffer [2-(N-morpholino)ethane sulfonic acid] pH 5.2 in the dark, was at a similar low level as in whole, darkened leaves. By addition of acetate or propionic acid, butyric acid or benzoic acid, NR was activated to or beyond the light level. The pH of crude tissue extracts was decreased by 0.5–1 pH units. Tissue acidification caused an inhibition of photosynthesis and of dark CO2 fixation. The acid-induced activation of NR in vivo was largely prevented by okadaic acid, an inhibitor of Type 1 and Type 2A protein phosphatases. This indicates that acid-induced activation was mediated by protein dephosphorylation. When, on the other hand, leaf discs were illuminated on Ches-buffer (2-[ N-cyclohexylamino]ethane sulfonic acid) pH 9 in the presence of bicarbonate (80 mM), their NR was as active as in intact leaves. Addition of ammonium chloride (up to 6 mM) caused a pH increase of the tissue extract up to 0.9 pH units. At the same time NR was inactivated to the dark level. Methionine sulfoximine did not prevent the ammonium effect. Photosynthesis and dark CO2 fixation were stimulated at pH 9 by ammonium chloride (1–2· mol· m −3) and were only slightly inhibited by up to 6 mol· m−3. The modulation of NR by acid-base treatment in vivo was fully reversible. The response of the NR system to acid or base treatment is consistent with a proposed role of nitrate reduction in the cellular pH-stat. The observation also indicates that cytosolic pH changes may be involved the signal chain triggering the modulation of NR.
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  • 2
    ISSN: 1432-2048
    Keywords: Key words: Activation state (nitrate reductase) ; Anoxia ; Hordeum (roots) ; Nitrate reductase ; Protein phosphory-lation ; Protein turnover
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract. The NADH-dependent nitrate reductase (NR, EC 1.6.6.1) in roots of hydroponically grown barley seedlings was extracted, desalted and the activity measured in buffer containing either Mg2+ (10 mM) or EDTA (5 mM). The former gives the actual NR activity (NRact) equivalent to dephospho-NR, whereas the latter gives the maximum NR capacity of the dephospho-form (NRmax). Both values together permit an estimation of the NR-phosphorylation state. Changes in NRact and NRmax were followed in response to root aeration or to shoot illumination or shoot removal, and were correlated with sugar contents and adenylate levels. Ethanol formation was also measured in roots differing in NR activity in order to obtain information on the relation between anaerobic alcoholic fermentation and nitrate reduction. In aerated roots, NR was highly phosphorylated (about 80%) and largely inactive. It was partly dephosphorylated (activated) by anoxia or by cellular acidification (pH 4.8 plus propionic acid). Anaerobic activation (dephosphorylation) of NR was stronger at acidic external pH (5) than at slightly alkaline pH (8), although ATP levels decreased and AMP levels increased at pH 5 and at pH 8 to the same extent. Thus, rapid changes in the NR-phosphorylation state in response to anaerobiosis were not directly triggered by the adenylate pool, but rather by cytosolic pH. Under prolonged darkness (24 h) or after shoot removal, NRmax decreased slowly without a large change in the phosphorylation state. This decrease of NRmax was correlated with a large decrease in the sugar content, and was prevented by glucose feeding, which had only minor effects on the phosphorylation state. Cycloheximide also prevented the decrease in NRmax without affecting the phosphorylation state. In contrast, anaerobiosis or cellular acidification prevented the decrease of NRmax and at the same time decreased the NR-phosphorylation state. It is suggested that NR turnover in roots is controlled by several factors: NR synthesis appears to depend on sugar availability, which has little effect on the phosphorylation state; in addition, NR degradation appears to be strongly affected by the phosphorylation state in such a way that the inactive phospho-NR is a better substrate for NR degradation than the dephospho-form. The rate of anaerobic ethanol formation was not affected by NR activity, indicating that the purpose of NR activation under hypoxia or anoxia is not to decrease or prevent alcoholic fermentation.
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  • 3
    ISSN: 1432-2048
    Keywords: Key words: Activation state (nitrate reductase) ; Hordeum (nitrate reductase) ; Nitrate reductase ; Nitrate supply ; Phosphate deficiency ; Signal metabolites
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract. The relation between nitrate reductase (NR; EC 1.6.6.1) activity, activation state and NR protein in leaves of barley (Hordeum vulgare L.) seedlings was investigated. Maximum NR activity (NRAmax) and NR protein content (Western blotting) were modified by growing plants hydroponically at low (0.3 mM) or high (10 mM) nitrate supply. In addition, plants were kept under short-day (8 h light/16 h dark) or long-day (16 h light/8 h dark) conditions in order to manipulate the concentration of nitrate stored in the leaves during the dark phase, and the concentrations of sugars and amino acids accumulated during the light phase, which are potential signalling compounds. Plants were also grown under phosphate deficiency in order to modify their glucose-6-phosphate content. In high-nitrate/long-day conditions, NRAmax and NR protein were almost constant during the whole light period. Low-nitrate/long-day plants had only about 30% of the NRAmax and NR protein of high-nitrate plants. In low-nitrate/long-day plants, NRAmax and NR protein decreased strongly during the second half of the light phase. The decrease was preceded by a strong decrease in the leaf nitrate content. Short daylength generally led to higher nitrate concentrations in leaves. Under short-day/low-nitrate conditions, NRAmax was slightly higher than under long-day conditions and remained almost constant during the day. This correlated with maintenance of higher nitrate concentrations during the short light period. The NR activation state in the light was very similar in high-nitrate and low-nitrate plants, but dark inactivation was twice as high in the high-nitrate plants. Thus, the low NRAmax in low-nitrate/long-day plants was slightly compensated by a higher activation state of NR. Such a partial compensation of a low NRmax by a higher dark activation state was not observed with phosphate-depleted plants. Total leaf concentrations of sugars, of glutamine and glutamate and of glucose-6-phosphate did not correlate with the NR activation state nor with NRAmax.
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  • 4
    ISSN: 1432-1963
    Keywords: Schlüsselwörter Chondroblastom ; Knochentumoren ; Immunhistologie ; Proliferation ; Key words Chondroblastoma ; Bone tumors ; Immunohistochemistry ; Proliferation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Description / Table of Contents: Summary Representing only about 1 % of all primary bone tumors, chondroblastoma constitutes a very rare bone tumor entity. 56 cases of chondroblastoma, that had been collected by the Hamburg Bone Tumor Registry from 1972 to 1995, were examined histologically together with the radiological and clinical findings. In addition immunohistochemistry with antibodies against S 100, PGM1, LCA and the proliferationmarker MIB 1 was performed. The mean age was 20.4 years and male patients being the majority with a gender ratio of 2.7 : 1. Predominant localisation was the epiphyses of the long bones, although almost 40 % of the tumors were located at untypical sites. Usually a well-circumscribed lysis could be seen on plain X-Ray examination, however partial cortical destruction could be observed in one third of the cases. Histologically characteristic was a polygonal cell component with a weblike chonroid matrix, sometimes with a plane-like appearance. 5 cases showed a distinct nuclear polymorphism making a distinction from osteosarcoma difficult. Using immunohistochemistry all tumors except for one showed positive reaction for S 100 protein. Although the histogenesis of chondroblastoma is not completely understood, morphological findings as well as the observed reactivity with the S 100 protein indicate the chondroid origin. No reactivity for PGM 1 (CD 68) or LCA could be detected. All chondroblastoma showed a low rate of proliferation, thereby being distinguishable from high malignant bone tumors. In general chondroblastoma show a benign biological behavior. Different behavior was observed in 2 cases. One relapse located in the pelvis revealed local aggressive growth while in another case in the humerus a malignant transformation had taken place.
    Notes: Zusammenfassung 56 Chondroblastome, die im Hamburger Knochentumorregister im Zeitraum von 1972 bis 1995 archiviert wurden, wurden retrospektiv histologisch untersucht, unter Berücksichtigung des radiologischen Befunds sowie der klinischen Angaben. Zusätzlich wurden immunhistologische Färbungen für S 100, PGM 1, LCA und den Proliferationsmarker MIB 1 durchgeführt. Das Durchschnittsalter der Patienten betrug 20,4 Jahre unter Bevorzugung männlicher Patienten mit einem Geschlechtsverhältnis von 2,7 : 1. Bevorzugter Lokalisationsort waren die Epiphysen der langen Röhrenknochen. Radiologisch stellt sich typischerweise eine umschriebene Lyse mit umgebendem Randsaum dar. Charakteristisch ist histologisch eine polygonale Zellkomponente mit einer netzartigen chondroiden Matrix. In 5 Fällen lag eine deutliche Kernpolymorphie vor, die eine Abgrenzung zum Osteosarkom schwierig machte. Immunhistologisch waren mit Ausnahme eines Falle alle Tumoren positiv für S 100. Allen Chondroblastomen war eine niedrige Proliferationsrate gemeinsam, die diese deutlich von hochmalignen Knochentumoren unterschied. Chrondroblastome besitzen üblicherweise ein gutartiges biologisches Verhalten. Zwei Fälle dieser Studie zeigten einen davon abweichenden Verlauf. Bei einem Rezidivtumor im Becken zeigte sich ein lokal aggressives Wachstum, in einem anderen Fall im Humerus war es zu einer malignen Transformation gekommen.
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  • 5
    ISSN: 1432-1963
    Keywords: Schlüsselwörter Chondrosarkom ; Knochentumoren ; Grading ; Morphometrie ; Histologie ; Key words Morphometry ; Bone neoplasma ; Chondrosarcoma ; Grading ; Histology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Description / Table of Contents: Summary Chondrosarcomas are frequent malignant bone tumors. Aside from different subtypes, such as dedifferentiated, mesenchymal and clear-cell chondrosarcoma, chondrosarcomas (classical chondrosarcoma) show different grades of differentiation. The borderline between chondroma and classical chondrosarcoma is not clearly defined. The same chondrosarcoma can be graded differently at different institutes. Standardized therapy concepts are currently in preparation. As the Hamburg Bone Tumor Registry is often consulted for chondrogenic tumors, the histological criteria are based on a series of 74 chondrosarcomas recorded there. The emphasis has been laid on a classification which can be used in daily routine and which is reproducible and in agreement with the classifications of other international groups. Grade I chondrosarcomas (50 %) can be distinguished only by growth criteria. The nuclei are small and show high chromatin density. Grade II chondrosarcomas (42 %) have medium-sized, regular nuclei with loose chromatin structure. The chondrocytes of grade III cases (8 %) show polymorphic nuclei. Binucleas forms, the number of mitoses and cellularity all show considerable overlap for all three grades. So far there are no immunohistological and molecular biological methods for reliable differentiation. The therapeutic consequences of the classification into grades are thorough curettage, in the case of grade I tumors, or complete resection, for grade II and III cases. The long-term results, however, need to be confirmed by a larger number of cases. From 1991 to 1995 the method was applied and proved to be easily practicable in daily diagnostic routine. Some 104 cases of classical chondrosarcomas (grade I 53 %, grade II 39 %, grade III 8 %) were analyzed. Two pathologists both assigned the same grade in 90 % of cases.
    Notes: Zusammenfassung Das Chondrosarkom gehört zu den häufigen malignen Knochentumoren. Neben unterschiedlichen Subtypen treten unterschiedliche Differenzierungsgrade des klassischen Chondrosarkoms auf. Die Übergänge von einem Chondrom, der benignen Variante chondrogener Tumoren, und dem klassischen Chondrosarkom sind histologisch fließend. Über die Bewertung der einzelnen Tumorformen liegen unterschiedliche Befunde vor. Einheitliche Therapiekonzepte werden derzeit erarbeitet. Deshalb werden die histologischen Kriterien an einer Serie von 74 Chondrosarkomen zusammengestellt. Dabei wird Wert auf eine in der täglichen Routine brauchbare und reproduzierbare Klassifikation gelegt. Grad-I-Chondrosarkome (50 %) können nur aufgrund von Wachstumskriterien von Chondromen unterschieden werden. Die Zellkerne sind klein, chromatindicht. Grad-II-Chondrosarkome (42 %) besitzen mittelgroße, gleichmäßige Zellkerne mit einem lockeren Chromatingerüst. Bei den Grad-III-Fällen (8 %) tritt eine Kernpolymorphie der Tumorchondrozyten auf. Sog. Doppelkernformen, die Zahl an Mitosen sowie die Zelldichte sind Kriterien mit einer großen Überschneidung in den 3 Differenzierungsgraden. Die therapeutischen Konsequenzen aus der Gradeinteilung sind entweder eine sorgfältige Kürettage bei Grad-I-Tumoren oder eine weite Resektion bei Grad-II- und Grad-III-Fällen. Die Langzeitergebnisse bedürfen allerdings noch einer Bewertung an größeren Kollektiven. Das Verfahren hat sich in der täglichen diagnostischen Praxis bei 104 Fällen mit einem klassischen Chondrosarkom als sehr gut anwendbar und reproduzierbar erwiesen.
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  • 6
    ISSN: 1432-1963
    Keywords: Schlüsselwörter Ki-67 ; MIB-1 ; Proliferation ; Osteosarkom ; Niedrig maligne Osteosarkome ; Knochentumoren ; Key words Ki-67 ; MIB-1 ; Proliferation ; Osteosarcoma ; Low grade osteosarcoma ; Bone tumors
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Description / Table of Contents: Summary Bone tumors represent a group of tumors of various dignity. In spite of this single tumor entities may display strong morphological resemblance to each other which can in turn result in profound difficulties in differential diagnosis. The biological behaviour of a tumor is mainly determined by its rate of proliferation. In this study the rate of proliferation of 64 bone tumors (30 high-grade central osteosarcomas, 6 low-grade osteosarcomas, 8 giant cell tumors, 8 aneurysmatic bone cysts, 5 osteoidosteomas/osteoblastomas, 7 fibrous dysplasias and 5 cases of a myositis ossificans) were analysed. Immunohistochemistry was performed on paraffin-embedded tissue sections using the MIB-1 monoclonal antibody. MIB-1 recognizes the proliferation-associated Ki-67 protein which is expressed during the active phases of the cell cycle but cannot be detected in senescent cells. Among high-grade central osteosarcomas a significantly higher rate of proliferation (average value 30 %) was found in comparison with low-grade osteosarcomas and other benign intraosseous bone tumors. This approach proved to be very useful in the distinction between high-grade and low-grade osteosarcomas as well as bone-forming intraosseous tumors. However distinguishing low-grade osteosarcomas from benign bone tumors by determining only the rate of proliferation was not possible, although interestingly, the proliferative rate of myositis ossificans, a purely reactive lesion, was in the range of the values determined for high-grade osteosarcoma.
    Notes: Zusammenfassung Knochentumoren stellen eine Gruppe von Tumoren mit unterschiedlicher Dignität dar. Trotzdem besteht zwischen den einzelnen Tumorentitäten z. T. eine starke morphologische Ähnlichkeit, die im Einzelfall zu erheblichen differentialdiagnostischen Schwierigkeiten führen kann. Da das biologische Verhalten eines Tumors wesentlich durch seine Wachstumsgeschwindigkeit bestimmt wird, wurde die Proliferation an 64 Knochentumoren (30 hochmaligne zentrale Osteosarkome, 6 niedrig maligne Osteosarkome, 8 Riesenzelltumoren, 8 aneurysmatische Knochenzysten, 5 Osteoid-Osteome/Osteoblastome, 7 fibröse Dysplasien) und an 5 Fällen einer Myositis ossificans immunhistologisch durch den monoklonalen Antikörper MIB-1 an Paraffinmaterial untersucht. MIB-1 erkennt das Ki-67-Protein, das in den aktiven Phasen des Zellzyklus exprimiert wird, in ruhenden Zellen aber nicht nachweisbar ist. Bei den hochmalignen zentralen Osteosarkomen fand sich eine hohe Proliferationsrate von durchschnittlich annähernd 30 %, die signifikant höher war als die der niedrigmalignen Osteosarkome und der übrigen benignen intraossären Knochentumoren. Eine Abgrenzung zwischen den niedrigmalignen Osteosarkomen und den benignen Knochentumoren war durch die Bestimmung der Proliferationsrate allerdings nicht möglich. Die Ermittlung der Proliferationsrate mit dem Antikörper MIB-1 ist hilfreich zur Abgrenzung hochmaligner Osteosarkome von niedrig malignen Osteosarkomen und benignen knochenbildenden intraossären Tumoren, innerhalb dieser Tumoren ist aber eine weitere Differenzierung mit Hilfe der Proliferationsrate nicht möglich. Eine Ausnahme bildet die Myositis ossificans, die zeigt, daß auch rein reaktive Läsionen eine Proliferationsrate besitzen können, die die Werte hochmaligner Tumore erreicht.
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    Planta 191 (1993), S. 173-179 
    ISSN: 1432-2048
    Keywords: Anaerobiosis ; Enzyme modulation ; Nitrate reductase ; Pisum ; Protein phosphorylation ; Root
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The regulatory properties of nitrate reductase (NR; EC 1.6.6.1) in root extracts from hydroponically grown pea (Pisum sativum L. cv. Kleine Rheinländerin) plants were examined and compared with known properties of NR from spinach and pea leaves. Nitrate-reductase activity (NRA) extracted from pea roots decreased slowly when plants were kept in the dark, or when illuminated plants were detopped, with a half-time of about 4 h (= slow modulation in vivo). In contrast, the half-time for the dark-inactivation of NR from pea leaves was only 10 min. However, when root tip segments were transferred from aerobic to anaerobic conditions or vice versa, changes in NRA were as rapid as in leaves (= rapid modulation in vivo). Nitrate-reductase activity was low when extracted from roots kept in solutions flushed with air or pure oxygen, and high in nitrogen. Okadaic acid, a specific inhibitor of type-1 and type-2A protein phosphatases, totally prevented the in vivo activation by anaerobiosis of NR, indicating that rapid activation of root NR involved protein dephosphorylation. Under aerobic conditions, the low NRA in roots was also rapidly increased by incubating the roots with either uncouplers or mannose. Under these conditions, and also under anaerobiosis, ATP levels in roots were much lower than in aerated control roots. Thus, whenever ATP levels in roots were artificially decreased, NRA increased rapidly. The highly active NR extracted from anaerobic roots could be partially inactivated in vitro by preincubation of desalted root extracts with MgATP (2 mM), with a half-time of about 20 min. It was reactivated by subsequently incubating the extracts with excess AMP (2 mM). Thus, pea root NR shares many of the previously described properties of NR from spinach leaves, suggesting that the root enzyme, like the leaf enzyme, can be rapidly modulated, probably by reversible protein phosphorylation/ dephosphorylation.
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  • 8
    ISSN: 1432-2048
    Keywords: Enzyme modulation ; Nitrate reductase ; Protein kinase ; Protein phosphorylation ; Protein purification ; Spinacia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Using a three-step purification procedure, two protein fractions which catalyzed the ATP-dependent in-activation of nitrate reductase (NR) were obtained from spinach (Spinacia oleracea L.) leaf extracts. Purification involved ammonium-sulfate fractionation, anion-exchange chromatography and size-exclusion chromatography. The capacity of the fractions to inactivate NR by preincubation with ATP was examined by using as target either a crude NR-ammonium sulfate precipitate or partially purified NR (ppNR). The fractions were also examined for protein-kinase activity by measuring the phosphorylation of histone III S (or casein) with γ-[32P]ATP as substrate, and subsequent SDS-PAGE, autoradiography and liquid scintillation counting of cut-off histone bands. The two proteins had apparent molecular weights in the 67-kDa and 100-kDa region (termed P67 and P100, respectively). Neither P67 nor P100 alone was able to inactivate ppNR by preincubation with ATP. However, when P100 and P67 were added together to ppNR, ATP-dependent inactivation was observed, with a half-time of about 10 min. The P67, but not P100 had histone-kinase activity (casein was not phosphorylated). Using the partially purified system, various compounds were examined as possible effectors of NR inactivation. Sugar phosphates had little effect on the inactivation of NR. Addition of AMP at very high concentrations (5 mM), and removal of Mg2+ by excess EDTA also prevented the inactivation.
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  • 9
    ISSN: 1432-2048
    Keywords: AMP ; Cation (divalent) ; Nitrate reductase ; Pisum ; Protein phosphatase ; Spinacia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Nitrate reductase in spinach (Spinacia oleracea L.) leaves was rapidly inactivated in the dark and reactivated by light, whereas in pea (Pisum sativum L.), roots, hyperoxic conditions caused inactivation, and anoxia caused reactivation. Reactivation in vivo, both in leaves and roots, was prohibited by high concentrations (10–30 μM) of the serine/threonine-protein phosphatase inhibitors okadaic acid or calyculin, consistent with the notion that protein dephosphorylation catalyzed by type-1 or type-2A phosphatases was the mechanism for the reactivation of NADH-nitrate reductase (NR). Following inactivation of leaf NR in vivo, spontaneous reactivation in vitro (in desalted extracts) was slow, but was drastically accelerated by removal of Mg2+ with excess ethylenediaminetetraacetic acid (EDTA), or by desalting in a buffer devoid of Mg2+. Subsequent addition of either Mg2+, Mn2+ or Ca2+ inhibited the activation of NR in vitro. Reactivation of NR (at pH 7.5) in vitro in the presence of Mg2+ was also accelerated by millimolar concentrations of AMP or other nucleoside monophosphates. The EDTA-mediated reactivation in desalted crude extracts was completely prevented by protein-phosphatase inhibitors whereas the AMP-mediated reaction was largely unaffected by these toxins. The Mg2+-response profile of the AMP-accelerated reactivation suggested that okadaic acid, calyculin and microcystin-LR were rather ineffective inhibitors in the presence of divalent cations. However, with partially purified enzyme preparations (5–15% polyethyleneglycol fraction) the AMPmediated reactivation was also inhibited (65–80%) by microcystin-LR. Thus, the dephosphorylation (activation) of NR in vitro is inhibited by divalent cations, and protein phosphatases of the PP1 or PP2A type are involved in both the EDTA and AMP-stimulated reactions. Evidence was also obtained that divalent cations may regulate NR-protein phosphatase activity in vivo. When spinach leaf slices were incubated in Mg2+ -and Ca2+-free buffer solutions in the dark, extracted NR was inactive. After addition of the Ca2+ /Mg2+-ionophore A 23187 plus EDTA to the leaf slices, NR was activated in the dark. It was again inactivated upon addition of divalent cations (Mg2+ or Ca2+). It is tentatively suggested that Mg2+ fulfills several roles in the regulatory system of NR: it is required for active NR-protein kinase, it inactivates the protein phosphatase and is, at the same time, necessary to keep phospho-NR in the inactive state. The EDTA- and AMP-mediated reactivation of NR in vitro had different pH optima, suggesting that two different protein phosphatases may be involved. At pH 6.5, the activation of NR was relatively slow and the addition or removal of Mg2+ had no effect. However, 5′-AMP was a potent activator of the reaction with an apparent K m of 0.5 mM. There was also considerable specificity for 5′AMP relative to 3′- or 2′-AMP or other nucleoside monophoposphates. We conclude that, depending upon conditions, the signals triggering NR modulation in vivo could be either metabolic (e.g. 5′-AMP) or physical (e.g. cytosolic [Mg2+]) in nature.
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  • 10
    ISSN: 1432-2048
    Keywords: Inhibitor protein ; Nitrate reductase ; Protein kinase ; Protein phosphatase ; Protein turnover
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Nitrate reductase activity and NR protein levels in various leaf tissues were drastically decreased (〈3.5% of normal activity) either by keeping detached leaves in continuous darkness for up to 6 d (spinach), or by growing plants (pea, squash) hydroponically on ammonium as the sole N-source, or by germinating and growing etiolated seedlings in complete darkness (squash). The presence of nitrate reductase protein kinase (NRPK), nitrate reductase protein phosphatase (NRPP) and inhibitor protein (IP) was examined by measuring the ability of NR-free desalted extracts to inactivate (ATP-dependent) and reactivate (5′-AMP/EDTA-dependent) added purified spinach NR in vitro. Extracts from low-NR plants (ammonium-grown pea and squash) were also prepared from leaves harvested at the end of a normal light or dark phase, or after treating leaves with anaerobiosis, uncouplers or mannose, conditions which usually activate NR in nitrategrown normal plants. Without exception, extracts from NR-deficient plant tissues were able to inactivate and reactivate purified spinach NR with normal velocity, irrespective of pretreatment or time of harvest. Considerable NRPK, NRPP and IP activities were also found in extracts from almost NR-free ripe fruits (cucumber and tomato). Activities were totally absent, however, in extracts from isolated spinach chloroplasts. The NRPK and IP fractions were partially purified with normal yields from NR-deficient squash or spinach leaves, following the purification protocol worked out for nitrate-grown spinach. The Ca2+/Mg2+-dependent kinase fraction from NR-deficient squash or spinach phosphorylated added purified spinach NR with γ-[32P]ATP and inactivated the enzyme after addition of IP. It is suggested (i) that the auxiliary proteins (NRPK, IP, NRPP) which modulate NR are rather species- or organ-unspecific, (ii) that they do not turn over as rapidly as does NR, (iii) that they are probably expressed independently of NR, and (iiii) that they are not covalently modulated, but under control of metabolic and/or physical signals which are removed by desalting.
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