RESUMO
To solve the problem of localization of the active center of cytochrome P-450 in microsomal membranes, new bifunctional compounds (I-IV), which contain pyridine radical, aliphatic chain of variable length and diphosphonic acid ("floating" molecules) have been applied. These compounds inhibit oxidation and binding of the substrates of cytochrome P-450 (aminopyrine and aniline), inhibition being of a competitive character. Measurements of distribution coefficients between water and membranes of microsomes and liposomes from egg phosphatidylcholine evidence that the microsomal proteins are necessary for providing effective interaction of I-IV with microsomal membrane. The 1H-NMR method has demonstrated compounds to be incorporated into lipid bilayer so that the non-polar part is in the inner membrane volume. The results obtained confirm our previous conclusion (Krainev A.G., Weiner L.M., Alferyev I.S., Slynko N.M. (1985) Biochim. Biophys. Acta, 818, 96-104) about localization of the active center of microsomal cytochrome P-450 at the depth of approximately 18 A from the hydrophilic surface of a membrane.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Microssomos Hepáticos/enzimologia , Aminopirina/metabolismo , Compostos de Anilina/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Fenômenos Químicos , Química , Cromatografia Líquida de Alta Pressão , Reagentes de Ligações Cruzadas/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , Inibidores das Enzimas do Citocromo P-450 , Membranas Intracelulares/enzimologia , Lipossomos/metabolismo , Espectroscopia de Ressonância Magnética , Oxirredução , Ratos , Ratos Endogâmicos , Espectrofotometria , Relação Estrutura-AtividadeRESUMO
A new approach, which we call 'float' molecules method for the determination of the active-centre location of cytochrome P-450 in a microsomal membrane, is proposed. We have synthesized new bifunctional compounds with the general formula: R-(CH2)n-S-CH2-CH(PO3H2)2, where n = 0,3,5,6,7,10 and R is a naphthalene-containing radical. The compounds inhibit oxidation and binding of cytochrome P-450 substrates of type I (naphthalene, aminopyrine) and of type II (aniline). The inhibition is of a competitive character. Compounds (I-V) neither affect NADPH-cytochrome c reductase, nor induce conversion of cytochrome P-450. A lipid-soluble fluorescent probe (1,6-diphenyl-1,3,5-hexatriene) has been used to show that these compounds do not affect melting of microsomal membrane. The 31P-NMR method has demonstrated compound (III) to be incorporated into microsomal membrane so that the hydrophilic part is in the water phase. The data obtained make it possible to estimate the distance (r) between the membranes surface and Fe3+ in the active centre of the enzyme (r less than or equal to 20 A) under the assumption that all molecules of cytochrome P-450 are equally remote from the membrane surface.
