Reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase and cytochrome b5 as electron carriers in NADH-supported cytochrome P-450 -dependent enzyme activities in liver microsomes |
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Authors: | E G Hrycay R A Prough |
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Affiliation: | Department of Biochemistry, The University of Texas Southwestern Medical School, Dallas, Texas 75235 U.S.A. |
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Abstract: | The role of NADH-cytochrome b5 reductase and cytochrome b5 as electron carriers in NADH-supported electron transport reactions in rat liver microsomes has been examined by measuring three enzyme activities: NADH-cytochrome P-450 reductase, NADH-peroxidase, and NADH-cytochrome c reductase. The first two reactions are known to involve the participation of an NADH-specific reductase and cytochrome P-450 whereas the third requires the reductase and cytochrome b5. Antibody prepared against NADH-cytochrome b5 reductase markedly inhibited the NADH-peroxidase and NADH-cytochrome c reductase activities suggesting the involvement of this NADH-specific reductase in these reactions. Liver microsomes prepared from phenobarbital-pretreated rats were digested with subtilisin to remove cytochrome b5 and the submicrosomal particles were collected by centrifugation. The specific content of cytochrome b5 in the digested particles was about 5% of that originally present in liver microsomes and all three enzyme activities showed similar decreases whereas NADH-ferricyanide reductase activity (an activity associated with the flavoenzyme NADH-cytochrome b5 reductase) remained virtually unchanged. Binding of an excess of detergent-purified cytochrome b5 to the submicrosomal particles at 37 °C for 20 min followed by centrifugation and enzymic measurements revealed a striking increase in the three enzyme activities. Further evidence for cytochrome b5 involvement in the NADH-peroxidase reaction was the marked inhibition by antibody prepared against the hemoprotein. These results suggest that in microsomal NADH-supported cytochrome P-450-dependent electron transport reactions, cytochrome b5 functions as an intermediate electron carrier between NADH-cytochrome b5 reductase and cytochrome P-450. |
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