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Biomedical subjects

B S Masters

Publications and source records attributed to B S Masters.

At least 127 records · Page 7Linked to original sources
PubMed1977

Evidence that puriified liver microsomal cytochrome P-450 is a one-electron acceptor.

Two forms of highly purified liver microsomal cytochrome P-450, P-450LM2, and P-450LM4, have been titrated with standardized solutions of sodium dithionite under anaerobic conditions. Only 1 electron was consumed per hemin molecule, and reoxidation of the reduced heme is accompanied by the transfer of 1 electron to oxidizing agents such as cytochrome c, cytochrome b5, or potassium ferricyanide. The present results are in disagreement with earlier dithionite titrations and reoxidation experiments which indicate that liver microsomal cytochrome P-450 is a 2-electron acceptor, but are in accord with previous potentimetric titrations and product yield data which indicate that this hemeprotein is a 1-electron acceptor. The cause for the discrepancy between the present and previous results is not fully understood.

Animals↗
PubMed1976

Some properties of a detergent-solubilized NADPH-cytochrome c(cytochrome P-450) reductase purified by biospecific affinity chromatography.

NADPH-cytochrome c (cytochrome P-450) reductase (EC 1.6.2.4) has been purified to homogeneity, as judged by sodium dodecyl sulfate disc gel electrophoresis, from detergent-solubilized rat and pig liver microsomes using an affinity chromatography procedure. Treatment of microsomes with a polyethoxynonylphenyl ether plus either cholate or deoxycholate and subsequent batch-wise DEAE-cellulose chromatography followed by biospecific affinity chromatography on Sepharose 4B-bound N6-(6-aminohexyl)-adenosine 2',5'-bisphosphate (2'5'-ADP-Sepharose 4B) result in a greater than 30% yield of purified reductase from microsomes. The enzyme contains 1 mol each of FAD and FMN and exhibits a molecular weight of 78,000 g mol-1 estimated by comparison with protein standards on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The turnover numbers calculated on the basis of flavin are 1360 min-1 and 1490 min-1 at 25 degrees for the pig and rat liver enzymes, respectively. Titration of these purified preparations aerobically with both NADPH and potassium ferricyanide demonstrated unequivocally that the air-stable, reduced form of NADPH-cytochrome c (P-450) reductase contains 2 electron equivalents, confirming recent results obtained by Masters et al. (Masters, B. S. S., Prough, R. A., and Kamin, H. (1975) Biochemistry 14, 607-613) for the proteolytically solubilized enzyme. In addition, these preparations are capable of reconstituting benzphetamine N-demethylation activity in the presence of partially purified cytochrome P-450 and dilauroylphosphatidylcholine, as measured by formaldehyde formation from benzphetamine.

Aerobiosis↗
PubMed1976

Mechanism of 2-naphthylamine oxidation catalysed by pig liver microsomes.

1. In pig liver microsomes 2-naphthylamine-dependent NADPH oxidation, oxygen reduction, and hydroxylamine formation are linear with time for several minutes. A sharp increase in NADPH oxidation and oxygen uptake then coincides with an abrupt loss of hydroxylamine from the medium. 2. The initial rate of 2-naphthylamine N-oxidation correlates with the microsomal concentration of mixed-function amine oxidase and the extent of linear accumulation of hydroxylamine is dependent on microsomal NADPH-cytochrome c reductase activity and concentration of lipid (microsomes). 3. Antisera to NADPH-cytochrome c reductase markedly decreased hydroxylamine accumulation during incubation but had no effect on the rate of 2-naphthylamine N-oxidation. 4. A system duplicating all of the kinetic properties of the microsomal 2-naphthylamine oxidase was constructed with two purified flavoproteins, (mixed-function amine oxidase and NADPH-cytochrome c reductase) and a lipid phase (erythrocyte ghosts or synthetic lecithin liposomes). 5. By independently varying the concentrations of each component in the reconstituted system, the contribution of each to the observed kinetics was defined. 6. In addition to the initial N-oxidation of 2-naphthylamine, at least six other reactions contribute to the kinetic patterns of 2-naphthylamine oxidation catalysed by the reconstituted system.

2-Naphthylamine↗
PubMed1976

The catalysis of heme degradation by purified NADPH-cytochrome C reductase in the absence of other microsomal proteins.

The report by Schacter et al. (J Biol Chem 247: 3601, 1972) that an antibody to NADPH-cytochrome c oxidoreductase inhibited NADPH-cytochrome c reductase and heme oxygenase activities in rat and pig liver and spleen microsomes demonstrated the role of this flavoprotein in microsomal heme oxygenation. Recent studies from other laboratories (Yoshida et al., J Biochem 75, 1187: 1974 and Bissell et al., Fed Proc 33: 1246, 1974) have strongly suggested that cytochrome P-450 is not involved in heme oxygenation. The availability of a homogeneous preparation of NADPH-cytochrome c reductase prompted us to test heme oxygenase activity in a system devoid of hemoprotein contamination. NADPH-cytochrome c reductase catalyzed biliverdin formation at a rate of 8.26 +/- 0.5 SEM nmole min-1mg-1 in the absence of biliverdin reductase. The rate of bilirubin formation in the presence of biliverdin reductase was less than 10% of the rate of biliverdin formation, suggesting that mixture of biliverdin isomers may be produced. Biliverdin production was potently (70--80%) inhibited by catalase, but was unaffected by superoxide dismutase. Epinephrine also inhibited heme oxygenation, presumably by utilizing O2. required for the formation of H2O2 by the reductase. By extrapolation, the NADPH oxidase activity due to NADPH-cytochrome c reductase can account for heme degradation occurring in microsomes. However, the specificity of ring scission at the IXalpha position must be due to another microsomal protein, perhaps the heme oxygenase of Yoshida et al., and not cytochrome P-450.

Animals↗