scholarly journals Rat hepatic microsomal cytochrome b5. A simple large-scale purification procedure and antibody production by antigen-containing liposomes

1988 ◽  
Vol 256 (3) ◽  
pp. 1051-1054 ◽  
Author(s):  
J Carlsen ◽  
K Christiansen ◽  
H M Jensen
Keyword(s):  
Gel Electrophoresis ◽  
Large Scale ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cytochrome B5 ◽  
Liver Microsomes ◽  
Lipid Vesicles ◽  
Rat Liver Microsomes ◽  
Native Form ◽  
Great Capacity ◽  
Microsomal Cytochrome B5

Cytochrome b5 from rat liver microsomes (microsomal fractions) was purified in its native form. The procedure described has great capacity, is fast, and the final product is pure as judged from SDS/polyacrylamide-gel electrophoresis. Antibodies to cytochrome b5 are obtained after administration of the antigen inserted into small unilamellar lipid vesicles.

scholarly journals A simple method for purification of rat hepatic microsomal cytochrome b5

1985 ◽  
Vol 226 (1) ◽  
pp. 339-341 ◽  
Author(s):  
A Burchell
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cytochrome B5 ◽  
Sodium Dodecyl ◽  
High Yield ◽  
Simple Method ◽  
Microsomal Cytochrome ◽  
Microsomal Cytochrome B5 ◽  
Polypeptide Band

Rat hepatic microsomal cytochrome b5 was purified to homogeneity by solubilization with the detergent Lubrol 12-A9 and chromatography on Fractogel TSK DEAE-650(S). The protein was obtained in high yield (52-87%) in 8 h, and only one polypeptide band, of Mr 16 600, was visible after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis.

scholarly journals Localization and biosynthesis of NADH-cytochrome b5 reductase, an integral membrane protein, in rat liver cells. II. Evidence that a single enzyme accounts for the activity in its various subcellular locations.

1980 ◽  
Vol 85 (3) ◽  
pp. 516-526 ◽  
Author(s):  
J Meldolesi ◽  
G Corte ◽  
G Pietrini ◽  
N Borgese
Keyword(s):  
Cytochrome C ◽  
Rat Liver ◽  
Reductase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cytochrome B5 ◽  
Liver Microsomes ◽  
Water Soluble ◽  
Rat Liver Microsomes ◽  
Cytochrome B5 Reductase ◽  
Nadh Cytochrome

NADH-cytochrome b5 reductases of rat liver microsomes, mitochondria, and heavy and light Golgi fractions (GF3 and GF 1+2) were compared by antibody inhibition and competition experiments, by peptide mapping, and by CNBr fragment analysis. The water-soluble portion of the microsomal enzyme, released by lysosomal digestion and purified by a published procedure, was used to raise antibodies in rabbits. Contaminant antimicrosome antibodies were removed from immune sera by immunoadsorption onto the purified antigen, and the F(ab')2 fragments of the pure antireductase antibody thus obtained were found to inhibit the NADH-cytochrome c reductase activity equally well in the four membrane fractions investigated, with similar dose-response relationships. Moreover, the purified water-soluble fragment of microsomal reductase, which by itself is very inefficient in reducing cytochrome c, competed for antibody binding with the membrane-bound enzymes, and therefore prevented the inhibition of their activity not only in microsomes but also in the other fractions. The reductases isolated from detergent-solubilized microsomes, mitochondria, GF3, and GF1+2 by immunoadsorption had identical mobilities in SDS polyacrylamide gels. The corresponding bands were eluted from gels, fragmented with pepsin or CNBr treatment, and the two families of peptides thus obtained were analyzed by two-dimensional mapping and SDS polyacrylamide gel electrophoresis, respectively. Both analyses failed to reveal differences among reductases of the four fractions. These findings support the hypothesis that NADH-cytochrome b5 reductase in its various subcellular locations is molecularly identical.

scholarly journals Analytical study of microsomes and isolated subcellular membranes from rat liver. VI. Electron microscope examination of microsomes for cytochrome b5 by means of a ferritin-labeled antibody.

1976 ◽  
Vol 71 (2) ◽  
pp. 551-564 ◽  
Author(s):  
J Remacle ◽  
S Fowler ◽  
H Beaufay ◽  
A Amarcostesec ◽  
J Berthet
Keyword(s):  
Electron Microscope ◽  
Rat Liver ◽  
Sucrose Gradient ◽  
Analytical Study ◽  
Cytochrome B5 ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Microscope Examination ◽  
Group B ◽  
Isopycnic Centrifugation

The distribution of cytochrome b5 in rat liver microsomes, and in two microsomal subfractions isolated by density equilibration in a linear sucrose gradient, was studied under the electron microscope by means of a ferritin-labeled hybrid anti-cytochrome b5/anti-ferritin antibody. Results of this study show that cytochrome b5 is present in essentially all microsomal vesicles derived from endoplasmic reticulum (ER), whether rough or smooth. Thus, the dissociation of ER constituents into two groups (b and c), achieved by subfractionating microsomes by isopycnic centrifugation (Beaufay, H., A. Amar-Costesec, D. Thines-Sempoux, M. Wibo, M. Robbi, and J. Berthet. 1974. J. Cell Biol. 61:213-231), does not reflect the association of each group with distinct microsomal particles but reflects rather an enzymatic heterogeneity of the ER: the ratio of group c to group b enzymes increasing with the density and ribosome load of the particles.

scholarly journals Characterization and biosynthesis of cytochrome b5 in rat liver microsomes

1968 ◽  
Vol 107 (6) ◽  
pp. 839-849 ◽  
Author(s):  
J. R. Sargent ◽  
B. P. Vadlamudi
Keyword(s):  
Amino Acid ◽  
Rat Liver ◽  
Spectral Properties ◽  
Proteolytic Enzymes ◽  
Cytochrome B5 ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Isolated Rat Liver ◽  
Peptide Linkage ◽  
Isolated Rat

1. Cytochrome b5 is released from rat liver microsomes by both proteolytic enzymes and by treatments that disrupt phospholipids. Cytochrome P-420 is only released to a marked extent by treatments that disrupt phospholipids. 2. Cytochrome b5 was isolated in a pure state from both the rough and smooth fractions of rat liver microsomes after treatment with trypsin, and was shown to contain two cytochrome components with identical spectral properties. 3. Amino acid analyses of the two components are presented, together with peptide ‘fingerprint’ patterns of tryptic digests of the two components. 4. Studies based on the direct isolation of cytochrome b5 after administration of a single dose of radioactive amino acid to rats demonstrate that the cytochrome is synthesized initially in the rough fraction of microsomes and only subsequently appears in the smooth fraction. 5. Isolated rat liver microsomes are capable of incorporating radioactive amino acids into cytochrome b5 under standard conditions. 6. Under these conditions the amino acid is incorporated into peptide linkage in the cytochrome.

Cytochrome b5/Cytochrome b5 Reductase Complex in Rat Liver Microsomes has NADH-Linked Aquacobalamin Reductase Activity

1992 ◽  
Vol 122 (4) ◽  
pp. 940-944 ◽  
Author(s):  
Fumio Watanabe ◽  
Yoshihisa Nakano ◽  
Hisako Saido ◽  
Yoshiyuki Tamura ◽  
Hiroyuki Yamanaka
Keyword(s):  
Rat Liver ◽  
Reductase Activity ◽  
Cytochrome B5 ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Cytochrome B5 Reductase

scholarly journals Absence of sugars in electrophoretically purified cytochrome b5 demonstrated by combined gas chromatography-mass spectrometry.

1981 ◽  
Vol 89 (3) ◽  
pp. 615-620 ◽  
Author(s):  
J Stadler
Keyword(s):  
Mass Spectrometry ◽  
Gas Chromatography ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cytochrome B5 ◽  
Sodium Dodecyl ◽  
Gas Chromatography Mass Spectrometry ◽  
Rat Liver Microsomes ◽  
Amino Terminal ◽  
Chromatography Mass Spectrometry ◽  
Sds Page

The problem of determining small but significant amounts of carbohydrates, in purified proteins, has been studied using the membrane protein, cytochrome b5. A newly developed method that involves direct gas chromatography-mass spectrometry of sugars obtained by hydrolysis of proteins purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) allows the identification and determination of small amounts of carbohydrates (e.g., 20 micrograms of glycoprotein containing a minimum of 0.1% monosaccharide), even in the presence of relatively high amounts of impurities. Application of this method to cytochrome b5 fragments obtained by tryptic digestion from rat liver microsomes and purified by combined gel filtration and ion exchange chromatography, followed by SDS PAGE, has consistently yielded values below 0.07 mol of the individual sugars and aminosugars per mole cytochrome b5. It is concluded that cytochrome b5, at least its trypsin-released major amino-terminal fragment, is not constitutively glycosylated.

scholarly journals Molecular and antigenic properties of cytochrome b5 from slow-muscle sarcoplasmic reticulum

1981 ◽  
Vol 197 (2) ◽  
pp. 515-518 ◽  
Author(s):  
G Salviati ◽  
S Salvatori ◽  
R Betto ◽  
A Margreth
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Reductase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cytochrome B5 ◽  
Slow Muscle ◽  
Molecular Weights ◽  
Antigenic Properties ◽  
Microsomal Cytochrome B5 ◽  
Membrane Bound ◽  
Nadh Cytochrome

NADH-cytochrome b5 reductase and cytochrome b5 associated with slow-muscle sarcoplasmic reticulum and liver microsomal fraction were identified with discrete protein bands of molecular weights 33000 and 16700 by polyacrylamide-gel electrophoresis. Purified detergent-extracted cytochrome b5 from muscle sarcoplasmic reticulum is indistinguishable from liver microsomal cytochrome b5 with respect to spectral properties, pI values and immunological reactivity with antibody to the liver cytochrome b5. Reaction of the antibody with membrane-bound cytochrome b5 inhibits the sarcoplasmic-reticulum NADH-cytochrome c reductase activity.

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