Binding of homogeneous cytochrome b5 to rat liver microsomes. Effect on N-demethylation reactions.
Incubation of rat homogeneous detergent-solubilized cytochrome b5 with rat liver microsomes resulted in specific binding of the hemoprotein which was rapidly reduced by NADH. The NADH cytochrome c reductase activity in these preparations increased in proportion to the amount of cytochrome bound. However, the extra-bound detergent-solubilized cytochrome b5 did inhibit NADPH-dependent N-demethylations, the NADH synergism and NADPH cytochrome P-450 reductase activity. Manganese protoporphyrin-apocytochrome complex when bound to microsomes in amounts equivalent to detergent-solubilised cytochrome b5 showed no effect on N-demethylation activity. Furthermore, the binding of cytochrome b5 preparations reconstituted from heme and apocytochrome b5 had no effect on either the NADPH-dependent N-demethylation of aminopyrene or ethylmorphine or the NADH synergism observed with rat liver microsomes. In addition, homogeneous cytochrome b5 eluted from three additional Sephadex G-100 columns showed no inhibitory effects when bound to liver microsomes. Spectral analyses of the acid-acetone extract of the hemoprotein showed an absorption peak at 278 nm suggesting that the homogeneous b5 contains contaminating amounts of tightly bound detergent which is responsible for the observed inhibition of mixed function oxidase activity and which is removed during extraction of the heme from the apocytochrome and during further gel filtration applications.
D000818:Animals; D001665:Binding Sites; D003579:Cytochrome Reductases / Q000378:metabolism; D003580:Cytochromes / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008345:Manganese / Q000494:pharmacology; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D013501:Surface-Active Agents / Q000494:pharmacology; D013696:Temperature
D003580:Cytochromes; D013501:Surface-Active Agents; D008345:Manganese; D010089:Oxidoreductases, N-Demethylating; D003579:Cytochrome Reductases