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Tissue-culture cell fractionation. Fractionation of cellular membranes from 125I/lactoperoxidase-labelled Lettree cells homogenized by bicarbonate-induced lysis: resolution of membranes by zonal centrifugation and in sucrose and metrizamide gradients.

ABSTRACT: 1. Lettrée cells were grown intraperitoneally in MF-1 mice and labelled extrinsically by the 125I/lactoperoxidase technique. 2. The cells were swollen in 1 mM-NaHCO3 and disrupted in a Dounce homogenizer. 3. Crude fractions of endoplasmic reticulum, plasma membrane and mitochondria were separated from a post-nuclear supernatant by sedimentation-rate gradient centrifugation in a BXIV zonal rotor. 4. Further resolution of these membranes was carried out in isopycnic sucrose gradients. 5. Bands of material from the latter were subfractionated in gradients of metrizamide. Some very pure subfractions of plasma membrane and endoplasmic reticulum were obtained. In addition, one subfraction containing 125I and NADPH-cytochrome c reductase but no Na++K+-stimulated adenosine triphosphatase and another containing these two enzymes but no 125I were resolved.

SUBMITTER: Graham JM

PROVIDER: S-EPMC1161246 | biostudies-other | 1979 Jul

REPOSITORIES: biostudies-other

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Tissue-culture fractionation. Zonal centrifugation of Lettree cells homogenized by glycerol-induced lysis: subfractionation of membranes in sucrose gradients.

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Tissue-culture cell fractionation. Fractionation of membranes from tissue-culture cells homogenized by glycerol-induced lysis.

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| S-EPMC1161244 | biostudies-other

The fractionation of nuclei from mammalian cells by zonal centrifugation.

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The characterization of rat kidney plasma membranes prepared by zonal centrifugation.

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| S-EPMC3553994 | biostudies-literature

Subcellular fractionation by differential and zonal centrifugation of aerobically grown glucose-de-repressed Saccharomyces carlsbergensis.

Project description:1. Homogenates were prepared from sphaeroplasts of aerobically grown glucose-de-repressed Saccharomyces carlsbergensis and the distributions of marker enzymes were investigated after differential centrifugation. Cytochrome c oxidase and cytochrome c were sedimented almost completely at 10(5)g-min, and this fraction also contained 37% of the catalase, 27% of the acid p-nitrophenyl phosphatase, 53 and 54% respectively of the NADH- and NADPH-cytochrome c oxidoreductases. 2. Zonal centrifugation indicated complex density distributions of the sedimentable portions of these enzymes and of adenosine triphosphatases and suggested the presence of two mitochondrial populations, as well as a bimodal distribution of peroxisomes and heterogeneity of the acid p-nitrophenyl phosphatase-containing particles. 3. Several different adenosine triphosphatases were distinguished in a post-mitochondrial supernatant that contained no mitochondrial fragments; these enzymes varied in their sensitivities to oligomycin and ouabain and their distributions were different from those of pyrophosphatase, adenosine phosphatase and adenosine pyrophosphatase. 4. The distribution of NADPH-cytochrome c oxidoreductase demonstrated that it cannot be used in S. carlsbergensis as a specific marker enzyme for the microsomal fraction. Glucose 6-phosphatase, inosine pyrophosphatase, cytochrome P-450 and five other enzymes frequently assigned to microsomal fractions of mammalian origin were not detected in yeast under these growth conditions.

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