The capacity of four native P-450 enzymes to interact with cytochrome b5 was compared and discussed in terms of spin shift and metabolism. Two-dimensional electrophoresis was employed as a tool to aid in characterization of the different enzymes isolated from liver microsomes of the untreated rat. RLM5 had a pl of 7.4 and RLM3 had a pl of 7.1. Two new forms isolated by this laboratory, RLM2 and RLM5a, differed from other forms of cytochrome P-450 characterized to date. The pl values of these forms were 7.35 and 7.6, respectively. The interaction of all four enzymes with cytochrome b5 differed. Cytochrome b5 caused a major low to high spin transition when added to RLM5. The latter hemoprotein was 28% high spin at 25 degrees C and was shifted to 55% high spin by cytochrome b5. RLM5a shifted from 4% high spin to 15% high spin under comparable conditions. In contrast, RLM2 and RLM3 were both minimally influenced by cytochrome b5, reaching only 8% high spin. Cytochrome b5 did not appreciably influence the rates of metabolism of aminopyrine, benzphetamine, testosterone, or p-nitroanisole with RLM2 or RLM3. However, with RLM5 and RLM5a, rates of aminopyrine and benzphetamine demethylation and testosterone hydroxylation were increased to about 130% with RLM5 and up to 200% with RLM5a. The demethylation of p-nitroanisole was stimulated by cytochrome b5, 3.5-fold with RLM5 and 14-fold with RLM5a. In no case was the ratio of monohydroxy metabolites of testosterone altered by the addition of cytochrome b5, indicating an effect on Vmax rather than Km.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMedDate not supplied

Influence of cytochrome b5 on the stoichiometry of the different oxidative reactions catalyzed by liver microsomal cytochrome P-450.

Stoichiometries of oxygen and NADPH consumption and product and hydrogen peroxide formation are examined for three forms of cytochrome P-450, LM2, RLM2, and RLM5, using several different substrates. As reported earlier, during the metabolism of some substrates [Gorsky, Koop, and Coon: J. Biol. Chem. 259, 6812-6817 (1984)], excess NADPH and oxygen are consumed suggesting that a 4-electron reduction of oxygen to water occurs. Similar effects are seen with testosterone as substrate for the constitutive forms of P-450, RLM2 and RLM5. However, when aminopyrine or p-nitroanisole serve as substrate, none of the forms of P-450 consumed excess NADPH or oxygen. Thus, consumption of excess NADPH and oxygen appears to be the result of the substrate used. Cytochrome b5 stimulates turnover of LM2 and RLM5 but not RLM2. With LM2, it causes a metabolic switching to occur between the monooxygenase and the NADPH-oxidase reaction. Although RLM5 is also stimulated by cytochrome b5, no metabolic switching occurs. Cytochrome b5 did not affect the proportion of excess NADPH or oxygen consumed.

Animals↗

PubMedDate not supplied

Stoichiometry of aminopyrine demethylation with and without NADH synergism.

The stoichiometry of aminopyrine demethylation by rat liver microsomes is examined. The stoichiometry corresponds with combined oxidase/mono-oxygenase activities, i.e., 1 NADPH:1 oxygen:1 product (HCHO + H2O2). NADH synergism of NADPH-supported aminopyrine demethylation is accompanied by a synergism of NADPH-supported hydrogen peroxide formation and a severalfold increase in NADH consumption, further suggesting a relationship between the oxidase and mono-oxygenase reactions. During synergism the rate of cytochrome b5 oxidation is increased severalfold over that when the hemoprotein is reduced in the presence of either pyridine nucleotide alone, in agreement with earlier reports. This increased rate of cytochrome b5 reoxidation approximates the sum of the increased rates of oxidase plus mono-oxygenase reactions (expressed in terms of reducing equivalents), but is less than the increased rate of NADH oxidation (also in reducing equivalents). The suggestion is made that the NADPH-supported reaction opens pathways of metabolism in which electrons from NADH can participate.

Aminopyrine↗