DIFFERENT INFLUENCES OF 2 FRACTIONS OF LUNG CYTOCHROME-B5 ON RECONSTITUTED LUNG BENZPHETAMINE N-DEMETHYLASE SYSTEM

Chromatography of lung microsomal cytochrome b5 obtained from DEAE-cel lulose columns, yielded two distinct cytochrome b5 fractions, These cy tochrome b5 fractions were further purified by Sephadex G-100 gel filt ration chromatography. The specific cytochrome b5 content of fraction 1 and fraction 2 was found to be 16.5 and 16.4 nmol/mg protein respect ively. Both fractions were free of cytochrome P-450, NADPH-cytochrome P-450 reductase and NADH-cytochrome b5 reductase activities. The effec ts of lung cytochrome b5 (fraction 1 and fraction 2) and liver cytochr ome b5 on benzphetamine N-demethylase activity were examined, Four dif ferent reconstitution systems were used, Lung cytochrome b5 fraction 2 and liver cytochrome b5 stimulated N-demethylase activity in all four systems when b5:P-450 molar ratio was low, i.e. 0.25 or 0.5, Both cyt ochrome b5 samples inhibited N-demethylase activity when b5:P-450 rati o exceeded 1:1 molar ratio, In contrast lung cytochrome b5 fraction 1 stimulated N-demethylase activity in all four systems, Maximal enhance ment of the activity was obtained when b5:P-450 ratio was 0.5. The gre atest increase in N-demethylation activity was observed in the reconst itution system with the lowest concentration of cytochrome P-450 reduc tase, conditions which most closely resemble intact microsomes.