Conversion Of Cytochrome P-450 Research Articles

Phospholipase A2-catalyzed conversion of hepatic cytochrome P450 to P420 in microsomal membranes prepared from young and old rats

Previous studies from this Laboratory have revealed few significant age-related changes in either hepatic microsomal cytochrome P450 content or NADPH-cytochrome P450 reductase activity, prompting the suggestion that the nearly ubiquitous age-associated decline in the hepatic metabolism of xenobiotics by the mixed-function oxidases is a result of alterations in the lipid matrix in which the macromolecular components are organized. In order to examine this postulate, the membrane microenvironment surrounding hepatic microsomal cytochrome P450 was enzymatically digested with snake venom phospholipase A2, and the kinetics of conversion of cytochrome P450 to cytochrome P420 determined for preparations obtained from rats of varying age. There were no significant differences in the kinetics of conversion of cytochrome P450 to cytochrome P420, suggesting that there are no significant age-related changes in the lipid microenvironment immediately adjacent to cytochrome P450. However, there was a slight difference, independent of age, in the rate of conversion of cytochrome P450 to cytochrome P420 between smooth and rough microsomes. The results are discussed in terms of general experimental approaches toward understanding drug metabolism in senescent organisms.

The Effects of Sulphydryl Reagents on the Binding and Mixed Function Oxidation of Hexobarbital in Rat Hepatic Microsomes

1. The effects of the sulphydryl reagents p-chloromercuribenzoate, N-ethylmaleimide and iodoacetamide on the binding spectrum, oxygen consumption and formation of a suspected substrate-cytochrome P-450-oxygen complex for hexobarbital in rat liver microsomes were investigated. 2. The oxygen consumption caused by hexobarbital oxidation was inhibited non-competitively by all three agents, with 50% inhibition at 4 times 10(-5) M for p-chloromercuribenzoate, 3-7 times 10(-4) M for N-ethylmaleimide and 1-9 times 10(-3) M for iodoacetamide. Cysteamine protected and at least partially reversed this inhibition. 3. p-chloromercuribenzoate inhibited the formation of the cytochrome P-450-substrate-oxygen complex, while N-ethylmaleimide and iodoacetamide also inhibited the formation of this complex but to a lesser extent. The p-chloromercuribenzoate inhibition was protected against and reversed by cysteamine. 4. p-Chloromercuribenzoate and N-ethylmaleimide caused a 50% reduction in the magnitude of the hexobarbital-induced binding spectrum, and this was paralleled by the conversion of cytochrome P-450 to cytochrome P-420. Cysteamine protected against this effect but could not reverse it. Iodoacetamide had no effect on the binding spectrum of hexobarbital and failed to convert cytochrome P-450 to cytochrome P-420. 5. Points of attack within the reaction sequence of drug oxidation are tentatively ascribed to the sulphydryl reagents used in this study.

Effects ofrypsin on the electron transport systems of liver microsomes.

Treatment of rat liver microsomes with low amounts of trypsin resulted in an inactivation of NADPH‐cytochrome P‐450 reductase parallel to the release of NADPH‐cytochrome c reductase. Associated herewith there was an inhibition of the NADPH‐linked drug hydroxylation and lipid peroxidation activities, as well as an inactivation of the reduction of cytochrome b5 by NADPH. The NADH‐cytochrome c reductase system, on the other hand, was considerably less sensitive to trypsin treatment of the microsomes. The inhibition of the NADH‐cytochrome c reductase activity at higher concentrations of trypsin was, however, likewise correlated with a solubilization of cytochrome b5. High concentrations of trypsin also caused a conversion of cytochrome P‐450 into cytochrome P‐420 and a disappearance of the spectral shift characteristic of the interaction of cytochrome P‐450 with substrates of the hydroxylating system.