Analytical Study of Microsomes and Isolated Subcellular Membranes From Rat Liver

Amar-Costesec, A; Wibo, Maurice; Thinessempoux, D.; Beaufay, Henri; Berthet, J

doi:10.1083/jcb.62.3.717

Cited by 215 publications

(122 citation statements)

References 32 publications

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“…These results are doubly meaningful, in that they show that the bulk of the microsomal cholesterol is physically independent from the structures bearing group b and ¢ enzymes, and also that the latter structures do not contain appreciable amounts of cholesterol per unit weight, at least in a form accessible to digitonin binding. Our conclusion, which is at variance with the views of other authors (13,23), will be further supported in the next paper of this series (5). The intracellular localization of cholesterol is examined below.…”

Section: Distribution Of Chemical Constituentscontrasting

confidence: 50%

“…Thus it is obvious that the rougher parts of the endoplasmic reticulum (ER) are essentially free of cholesterol. This conclusion may be extended to the remainder of the ER in view of the results of experiments (5,40) which have shown that previous treatment of the microsomes with small amounts of digitonin causes a considerable shift to higher densities of the distribution patterns of cholesterol and of some group a enzymes, without at all affecting those of group b and c enzymes. These results are doubly meaningful, in that they show that the bulk of the microsomal cholesterol is physically independent from the structures bearing group b and ¢ enzymes, and also that the latter structures do not contain appreciable amounts of cholesterol per unit weight, at least in a form accessible to digitonin binding.…”

Section: Distribution Of Chemical Constituentsmentioning

confidence: 98%

“…Analytical techniques (8) and the preparation and the composition of the microsomal fractions (4) have been described in previous papers of this series. Subsequent ones will report on the effects of various treatments of the microsomes (5). Some of the findings made in these experiments have since been reported at a symposium (2), as part of other publications (1,7,18,39,40,44), and in abstract form (3).…”

Section: Introductionmentioning

confidence: 99%

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Analytical Study of Microsomes and Isolated Subcellular Membranes From Rat Liver

Beaufay

Amar-Costesec

Thinessempoux

et al. 1974

The Journal of Cell Biology

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237

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Rat liver microsomal fractions have been equilibrated in various types of linear density gradients. 15 fractions were collected and assayed for 27 constituents. As a result of this analysis microsomal constituents have been classified, in the order of increasing median density, into four groups labeled a, b, c, and d. Group a includes: monoamine oxidase, galactosyltransferase, 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase, and cholesterol; group b: NADH cytochrome c reductase, NADPH cytochrome c reductase, aminopyrine demethylase, cytochrome b 5, and cytochrome P 450; group c: glucose 5-phosphatase, nucleoside diphosphatase, esterase, j3-glucuronidase, and glucuronyltransferase; group d: RNA, membrane-bound ribosomes, and some enzymes probably adsorbed on ribosomes: fumarase, aldolase, and glutamine synthetase. Analysis of the microsomal fraction by differential centrifugation in density gradient has further dissociated group a into constituents which sediment more slowly (monoamine oxidase and galactosyltransferase) than those of groups b and c, and 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase, and the bulk of cholesterol which sediment more rapidly (group a2). The microsomal monoamine oxidase is attributed, at least partially, to detached fragments of external mitochondrial membrane. Galactosyltransferase belongs to the Golgi complex. Group a2 constituents are related to plasma membranes. Constituents of groups b and c and RNA belong to microsomal vesicles derived from the endoplasmic reticulum. These latter exhibit a noticeable biochemical heterogeneity and represent at the most 80% of microsomal protein, the rest being accounted for by particles bearing the constituents of groups a and some contaminating mitochondria, lysosomes, and peroxisomes. Attention is called to the operational meaning of microsomal subfractions and to their cytological complexity.

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Section: Distribution Of Chemical Constituentscontrasting

confidence: 50%

Section: Distribution Of Chemical Constituentsmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Analytical Study of Microsomes and Isolated Subcellular Membranes From Rat Liver

Beaufay

Amar-Costesec

Thinessempoux

et al. 1974

The Journal of Cell Biology

Self Cite

237

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“…The faster moving band of the cytochrome b~ and heme preparations showed a green-yellow color, and did not bind the dye. resuspended in a 43.1% sucrose solution, z To increase the equilibrium density of contamination fragments of plasma membranes, digitonin was added in an amount equivalent to 0.135 mg/g fresh weight of liver originally homogenized (3). This and all subsequent sucrose solutions contained 3 mM imidazole-HC1 buffer, pH 7.4.…”

Section: Preparation Of Subcellular Membrane Fractionsmentioning

confidence: 99%

Analytical study of microsomes and isolated subcellular membranes from rat liver. V. Immunological localization of cytochrome b5 by electron microscopy: methodology and application to various subcellular fractions.

Fowler¹,

Remacle²,

Trouet³

et al. 1976

The Journal of Cell Biology

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The localization of cytochrome b5 on the membranes of various subcellular organelles of rat liver was studied by a cytoimmunological procedure using anticytochrome bdanti-ferritin hybrid antibodies and ferritin as label. For this study, highly purified and biochemically characterized membrane preparations were employed. Outer mitochondrial membranes were found to be heavily labeled by the hybrid antibodies whereas Golgi and plasma membranes were not marked by the reagent. Peroxisome membranes were moderately labeled by the hybrid antibodies, suggesting that they may contain some cytochrome bs. The preparation and purification of hybrid antibodies without peptic digestion is described and an analysis made of the composition of the final reagent product.Cytochrome b5 was first found in the microsomal fraction of rat liver (10, 45) and later shown to be associated with outer membranes of mitochondria (17,33,43). This protein has also been reported to be present in the Golgi membranes (8,16,21) and NADH: cytochrome b5 reductase activity is said to be present in peroxisomes (9, 13). On the basis of these data, cytochrome bs would appear to be present in membranes of several of the organelles of the liver cell. However, in these biochemical studies it is not always certain that the cytochrome b~ detected is truly associated with the organelle under study rather than being merely present as a subcellular contaminant of the preparations analyzed. In this paper, we report a sensitive ferritin-hybrid antibody technique that does permit, by morphological analysis, the specific localization of cytochrome b5 to individual membrane elements. In the course of developing this assay, we studied in detail the various steps in the preparation of our morphologic label, an antigenantibody complex between ferritin and the anticytochrome bdanti-ferritin hybrid antibody (abd aF). 1 We describe here our experience concerning 1 Abbreviations used in this paper: abdaF-ferritin, antigen-antibody complex between ferritin and the anti-cytochrome bs/anti-ferritin hybrid antibody; anti-X, an immunoglobulin or a fragment of an immunoglobulin, the specificity of which is undefined; p, density; ER, endoplasmic reticulum; PBS, 0.01 M Na phosphate, pH 7.4, in 0.15 M NaCI; SDS, sodium dodecyl sulfate.

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“…Although different membranes may be functionally related, they have been shown to be biochemically distinguishable entities in various systems [1][2][3][4]. Some enzymes, however, have been demonstrated to be localized in more than one type of membrane.…”

Section: Introductionmentioning

confidence: 99%

Immunological similarity of the NADH—cytochrome c electron transport system in microsomes, Golgi complex and mitochondrial outer membrane of rat liver cells

Borgese

Meldolesi

1976

FEBS Letters

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Analytical Study of Microsomes and Isolated Subcellular Membranes From Rat Liver

Cited by 215 publications

References 32 publications

Analytical Study of Microsomes and Isolated Subcellular Membranes From Rat Liver

Analytical Study of Microsomes and Isolated Subcellular Membranes From Rat Liver

Analytical study of microsomes and isolated subcellular membranes from rat liver. V. Immunological localization of cytochrome b5 by electron microscopy: methodology and application to various subcellular fractions.

Immunological similarity of the NADH—cytochrome c electron transport system in microsomes, Golgi complex and mitochondrial outer membrane of rat liver cells

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