Y. Yasukochi

Radical mechanism of aminopyrine oxidation by cumene hydroperoxide catalyzed by purified liver microsomal cytochrome P-450☆

Abstract Under identical experimental conditions, purified preparations of rabbit liver microsomal cytochrome P -450 and beef heart metmyoglobin were equally effective at stimulating the oxidation of aminopyrine to a free radical species by cumene hydroperoxide. Mannitol had no effect on radical levels produced with either hemeprotein-hydroperoxide system; however, specific ligands of the two hemeproteins, substrates of cytochrome P -450, and phospholipid affected the two systems quite differently. Only the metmyo-globindependent oxidation of aminopyrine was significantly inhibited by fluoride and cyanide. Metyrapone, a specific ligand of cytochrome P -450, and benzphetamine, which was N -demethylated by cumene hydroperoxide only in the presence of cytochrome P -450, inhibited only the cytochrome P-450 -stimulated oxidation of aminopyrine. Moreover, only with the solubilized liver hemeprotein was aminopyrine radical generation markedly stimulated by phospholipid. Similar properties of aminopyrine N -demethylation and radical formation by the cytochrome P-450 -cumene hydroperoxide system have strongly implicated the radical as a requisite intermediate in product formation. Micromolar concentrations of metyrapone caused parallel inhibition, by at least 50%, of both radical generation and formaldehyde production. These results support a radical pathway of N -demethylation proposed for other hemeprotein-hydroperoxide systems ( B. W. Griffin and P. L. Ting, 1978 , Biochemistry , 17 , 2206–2211), in which the substrate undergoes two successive one-electron abstractions, followed by hydrolysis of the iminium cation intermediate. Thus, for this class of substrates, the experimental data are consistent with the oxygen atom of the product arising from H 2 O and not directly from the hydroperoxide, which has been previously proposed as a general mechanism for cytochrome P -450 peroxidatic activities.

Comparison of the properties of detergent-solubilized NADPH-cytochrome P-450 reductases from pig liver and kidney. Immunochemical, kinetic, and reconstitutive properties.

Abstract NADPH-cytochrome P -450 reductases from pig liver and kidney and rabbit liver microsomes were purified to a specific activity of 50–62 μmol cytochrome c reduced/min/mg. All reductase preparations were separated into one major and one minor fraction on Sephadex G-200 columns. The molecular weights of the major fractions of the reductases were estimated to be 74,000, 75,000, and 75,500 for rabbit liver, pig kidney, and liver reductases, respectively, whereas the molecular weight of the minor fractions of these reductases, 67,000, was the same as that of the steapsin-solubilized pig liver reductase on SDS-polyacrylamide gel electrophoresis. K m values for NADPH and cytochrome c were: 20 and 29 μ m or 14 and 28 μ m for the pig kidney or liver reductase, respectively. Immunochemical studies, including Ouchterlony double diffusion experiments and inhibition of benzphetamine N -demethylation activity in microsomes by antibody against pig liver NADPH-cytochrome P -450 reductase, indicated the similarity of the purified liver and kidney reductases. There were no differences in the ability to reconstitute NADPH-mediated benzphetamine N -demethylation and laurate hydroxylation in reconstituted systems between the pig liver and kidney reductases, indicating that the reductase did not determine substrate specificity in these systems.

LAURATE HYDROXYLATION AND DRUG METABOLISM IN PIG LIVER AND KIDNEY MICROSOMES AND IN RECONSTITUTED SYSTEMS FROM PIG LIVER AND KIDNEY

Publisher Summary This chapter discusses laurate hydroxylation and drug metabolism in pig liver and kidney microsomes, and in reconstituted systems from pig liver and kidney. The study of monooxygenase systems in hepatic and extrahepatic tissues has necessitated the use of a variety of substrates to obtain measurable rates of metabolism. This situation suggests that there may be cytochromes P-450 in various organs with differing substrate specificities and physico-chemical properties. The metabolism of lauric acid to ω- and (ω-l)-hydroxylated products was described in rat liver microsomes and it was later shown to proceed at similar specific activities in rat kidney microsomes. Cytochrome P-450 was implicated as the terminal oxygenase in these systems. The physiological significance of fatty acid hydroxylation in liver is a subject of controversy, but the fact remains that it is an active substrate for liver and kidney microsomal metabolism. The chapter presents the studies in which lauric acid has been used in addition to other substrates to differentiate between two cytochromes P-450 Antibodies prepared to these cytochromes P-450 further substantiate their immunochemical non-identity and substrate specificity.

Studies on liver and kidney drug and lauric acid monooxygenases in both microsomal and reconstituted systems

Publisher Summary This chapter presents the studies on liver and kidney drug, and lauric acid monooxygenases in microsomal and reconstituted systems. The liver microsomal monooxygenase system metabolizes a broad spectrum of substrates, including xenobiotics, steroids, and fatty acids. However, the kidney microsomal monooxygenase system, while containing the same components as the liver system, metabolizes fatty acids at a higher specific activity than any other known substrate. The chapter describes a comparative study of the reductases and cytochromes P-450 of pig liver and kidney microsomes that was carried out to elucidate the differences in substrate specificity between the two organs. In this study, the purified kidney cytochrome P-450 from untreated pigs was found to be immunochemically distinct from the purified liver cytochrome P-450 from phenobarbital (PB)–induced pigs. In addition, differences between these purified cytochromes P-450 were found in benzphetamine demethylation and laurate hydroxylation activities measured in reconstituted systems. The two cytochromes P-450 that were isolated from liver microsomes of PB-induced pigs and from kidney microsomes of untreated pigs show no immunochemical cross-reactivity. They exhibit distinct substrate specificities that are affected neither by lipid milieu nor by the source of nicotinamide adenine dinucleotide phosphate-oxidase (NADPH)–cytochrome P-450 reductase.