Analytical Study of Microsomes and Isolated Subcellular Membranes From Rat Liver

Amar-Costesec, A; Beaufay, Henri; Wibo, Maurice; Thinessempoux, D.; Feytmans, Ernest; Robbi, Mariette; Berthet, J

doi:10.1083/jcb.61.1.201

Cited by 294 publications

(105 citation statements)

References 47 publications

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“…The relative specific activity of galactosyltransferase was 63 in the Golgi fraction, which was 70-75% pure and contained 10-15% protein belonging to ER components. The cytochrome bs content of the Golgi fraction was found to be 0.9/zg/ mg protein, which is about the amount expected to be present from contaminating ER components (2). Contamination of the peroxisome preparation was about 10% by ER and 6% by mitochondria.…”

Section: Ultrastructural Localization Of Cytochrome B5mentioning

confidence: 80%

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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Section: Ultrastructural Localization Of Cytochrome B5mentioning

confidence: 80%

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 microsomal subcellular fraction was prepared by differential centrifugation as previously described [18]. Microsomal protein concentration was measured as described by Lowry et al [19].…”

Section: Human Liver Microsomal Subcellular Fraction Preparationmentioning

confidence: 99%

Study of the B cell response to cytochrome P450IID6 in sera from chronic hepatitis C patients

Parez

Herzog

Jacqz-Aigrain

et al. 1996

Clinical and Experimental Immunology

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SUMMARYSome patients with chronic hepatitis C develop liver/kidney microsome type 1 antibodies. Some of these autoantibodies are directed against the cytochrome P450IID6. The aim of this study was to characterize the immunogenic sites on the P450IID6 molecule recognized by autoantibodies from individuals infected with the hepatitis C virus. Serum from 24 patients with markers of hepatitis C infection and liver/kidney microsome type 1 antibodies were tested by immunoblotting with human liver microsomal proteins. Eight of them with anti-P450IID6 antibodies were tested with synthetic peptides representing P450IID6 putative immunogenic sites and for their capacity to inhibit in vitro P450IID6 enzymatic activity. Anti-P450IID6 antibodies in the sera of chronic hepatitis C patients were of IgG subclass 1 and in one patient also of IgM type. These sera recognized different P450IID6 peptide sequences consisting of amino acids (aa) 200-214 and aa 271-339. The synthetic peptide between aa 321 and 339 appears to represent the main antigenic site. Five out of eight anti-P450IID6 positive sera were also able to inhibit P450IID6 enzymatic activity in vitro at a dilution of 1 : 100. The anti-P450IID6 autoimmune response in chronic hepatitis C patients is polyclonal, probably inducible, and maintained by the liberation of P450IID6 by hepatocyte lysis. The characterization of the P450IID6 immunogenic site recognized by patients with hepatitis C infection may enable a specific test to be designed to identify those patients with autoimmune hepatitis type 2.

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“…The state of this subject and the relevant contributions of other laboratories are summarized in the introductory paper of this series (6) which also justifies our approach to the question and describes the biochemical methods used. The procedure followed to prepare the microsome fraction and the biochemical properties of our microsomes were presented by Amar-Costesec et al (4), whereas morphometric data were reported by Wibo et al (33). Finally, a classification of several enzymic and chemical constituents t of rat liver microsomes into several groups was proposed according to both their distribution after density equilibration in various gradients and differential sedimentation in a shallow stabilizing gradient of sucrose (3,7).…”

Section: Introductionmentioning

confidence: 99%

“…All of these constituents equilibrated in the low density range (I.08 1.20) and were distinguished into those which sedimented faster (group a2: 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase, and cholesterol) than the bulk of microsomes, and those which sedimented more slowly (monoamine oxidase and galactosyltransferase). On the basis of the results presented here, and briefly alluded to before (7), a further distinction was proposed between monoamine oxi-1 As in the preceding papers (4,6,7), the term component designates a structural entity in intact cells or in tissue homogenates (for instance rough ER and rough microsomes), and the term constituent designates a biochemical entity (enzyme, cholesterol...). A group is a set comprising one or several constituents which behave similarly in all the fractionation systems used and can be distinguished from other constituents in at least one fractionation system (see the note added in proof, reference 7).…”

Section: Introductionmentioning

confidence: 99%

Analytical Study of Microsomes and Isolated Subcellular Membranes From Rat Liver

Amar-Costesec

Wibo

Thinessempoux

et al. 1974

The Journal of Cell Biology

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215

112

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lsopycnic equilibration and sedimentation rate studies of rat liver microsomes led previously to the assignment of microsomal constituents into group al (monoamine oxidase), group a2 (5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase and cholesterol), group a3 (galactosyltransferase), group b (NADH cytochrome ¢ reductase, NADPH cytochrome c reductase, aminopyrine demethylase, cytochrome b5 and P 450), and group c (glucose 6-phosphatase, esterase, nucleoside diphosphatase, 13-glucuronidase and glucuronyltransferase).Confirmation and extension of the assignment into groups has been obtained by studying the differential effect of the reagents digitonin, EDTA, and PPi. Digitonin specifically affected the equilibrium density only of the group a2 and (to a lesser extent) group a3, and not of groups b and c under conditions which preserved the structure-linked latency of nucleoside diphosphatase and galactosyltransferase. Within experimental error the rate of sedimentation of all microsomal constituents was unaffected. The morphological appearance under the electron microscope was indistinguishable from that of nondigitonin-treated microsomes, except that a few smooth membranes (< 10%) exhibited broken-looking profiles.Treatment of microsomes with EDTA or PPi detached a substantial part of RNA and released protein in excess over the amount accountable for by detachment of ribosome constituents. This detachment was confirmed by electron microscopy. EDTA and PPi decreased markedly the equilibrium density and the density dispersion of groups b and c, due mainly to the uncoating of rough

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Analytical Study of Microsomes and Isolated Subcellular Membranes From Rat Liver

Cited by 294 publications

References 47 publications

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.

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.

Study of the B cell response to cytochrome P450IID6 in sera from chronic hepatitis C patients

Analytical Study of Microsomes and Isolated Subcellular Membranes From Rat Liver

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