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  • 1
    Electronic Resource
    Electronic Resource
    Isolation and ultrastructural identification of membranes from the fungusGilbertella persicaria (1982)
    Springer
    Protoplasma 111 (1982), S. 87-106 
    Details
    ISSN: 1615-6102
    Keywords: Fungi ; Gilbertella persicaria ; Membranes ; Mitochondria ; Organelle isolation ; Plasma membrane ; Ultrastructure ; Vacuoles
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Methods are described for isolating and identifying subcellular membranes from walled hyphae ofGilbertella persicaria. Differences in thickness and symmetry of membranes and in contents of vesicles were used to distinguish different types of membranes. Mitochondria, vacuoles, plasma membrane, and vesicles with attached ribosomes from homogenized germlings equilibrated at the 1.2/1.4 M interface in discontinuous sucrose gradients. Accelerated flotation in centrifuged Ficol-sucrose gradients resulted in the additional separation of the mixed membranes into three fractions: one contained predominantly intact mitochondria, another was composed of vacuoles and vesicles coated with ribosomes, and a third was enriched in plasma membranes. Based upon morphometric analysis, these fractions contained 92% mitochondria, 53% vacuoles, and 89% plasma membranes, respectively. The source of vesicles coated with ribosomes was investigated since rapidly growing hyphae ofG. persicaria contained little rough endoplasmic reticulum as compared with other classes of membranes. Reconstruction from electron micrographs of mitochondrial fragmentation and vesiculation suggested that most of the ribosome-coated vesicles originated from disrupted mitochondria rather than from rough endoplasmic reticulum. The study demonstrates the utility of ultrastructural markers to identify membranesin vitro independent of, or as an adjunct to, cytochemical and biochemical markers.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01282067
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  • 2
    Electronic Resource
    Electronic Resource
    Sedimentation properties of chitosomes fromMucor rouxii (1984)
    Springer
    Protoplasma 122 (1984), S. 178-190 
    Details
    ISSN: 1615-6102
    Keywords: Chitin synthetase localization ; Chitosome sedimentation ; Fungus ; Miniorganelles ; Mucor rouxii ; Plasma membrane
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary This study was undertaken to assess the distribution and localization of chitin synthetase in a fungal cell and to evaluate the sedimentation behavior of chitosomes (microvesicular containers of chitin synthetase). Chitosomes were isolated from cell-free extracts of yeast cells ofMucor rouxii by rate-zonal and isopycnic sedimentation in sucrose density gradients. Because of their small size and low density, chitosomes were effectively separated from other subcellular particles. Rate-zonal sedimentation was a suitable final step for isolating chitosomes as long as ribosomes had been eliminated by enzymic digestion. By isopycnic centrifugation, chitosomes could be separated directly from a crude cell-free extract; they cosedimented with a sharp symmetrical peak of chitin synthetase at a buoyant density of d=1.14–1.15g/cm3; the only significant contaminants were particles of fatty acid synthetase complex. From such sedimentations, we estimated that 80–85% of the chitin synthetase activity in the cell-free extract was associated with chitosomes; the rest was found in two smaller peaks sedimenting at d=1.19–1.20 and d=1.21–1.22 (5–10%), and in the cell wall fraction (5–10%). By consecutive rate-zonal and isopycnic sedimentations, chitosome preparations with relatively few contaminating particles were obtained. Potassium/sodium phosphate buffer (pH 6.5)+MgCl2 was the most effective isolation medium for chitosomes. Other buffers such as TRIS-MES+MgCl2 led to massive aggregation of chitosomes and a change in sedimentation properties. This tendency of chitosomes to aggregate could explain why most of the chitin synthetase activity of a fungus is sometimes found associated with other subcellular structures,e.g., plasma membrane.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01281695
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    Paper (German National Licenses)
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