The reconstitution of microsomal redox chains. A comparitive analysis of the effectiveness of membrane self-assembly and template binding of electron carriers.
A comparative analysis was made of the effectiveness of three methods for the reconstitution of microsomal electron-transfer chains, namely, self-assembly, incorporation of electron carriers into liposomes (non-specific template) and incorporation into ;ghosts' of microsomal vesicles (specific template). It was shown that when the ;ghosts' of the microsomal vesicles were used as a specific template extra cytochrome b(5) and NADH-specific flavoprotein were incorporated into them, but cytochrome P-450 and NADPH-specific flavoprotein were not incorporated into the membrane. As a result of the self-assembly and incorporation into liposomes all the electron carriers were present in the reconstituted membrane. Cytochrome P-450 reactivation took place and the inactive form, cytochrome P-420, was converted into the active form, cytochrome P-450. Of the four enzyme hydroxylation systems studied, i.e. NADPH- and NADH-dependent p-hydroxylation of aniline, and NADPH- and NADH-dependent N-demethylation of dimethylaniline, only the NADH-dependent demethylation of dimethylaniline (60% of the initial value) and NADH-dependent p-hydroxylation of aniline (30% of the initial value) were reconstituted by self-assembly. NADPH oxidase and NADH oxidase activities were only properly reconstituted by self-assembly and incorporation into liposomes. In contrast, the NADPH-specific system of peroxidation of unsaturated fatty acids was reconstituted by specific template-binding.
D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D003580:Cytochromes / Q000378:metabolism; D004579:Electron Transport; D005420:Flavoproteins; D066298:In Vitro Techniques; D007700:Kinetics; D008081:Liposomes; D008297:Male; D008566:Membranes / Q000378:metabolism; D008854:Microscopy, Electron; D008861:Microsomes / Q000378:metabolism* / Q000648:ultrastructure; D009243:NAD; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009249:NADP; D010084:Oxidation-Reduction; D011485:Protein Binding; D051381:Rats
D003580:Cytochromes; D005420:Flavoproteins; D008081:Liposomes; D009243:NAD; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D009247:NADH, NADPH Oxidoreductases