Abstract
Several mono-, di- and trivalent cations were tested for effects on reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase (P-450 reductase) activity of rat liver microsomes. Most divalent cations (except heavy metals) stimulated P-450 reductase whereas mono- and trivalent ions had little effect or were inhibitory. The pH optimum of P-450 reductase was studied with microsomes from control, 3-methylcholanthrene-pretreated or phenobarbital-pretreated rats in the presence and absence of Mg++ and/or type I (benzphetamine) or type II (aniline) substrates or ligands. The pH optima curves were broad in all cases, but the action of effectors on P-450 reductase especially in microsomes from phenobarbital-pretreated rats seemed maximal in the pH 7.4 to 8.0 range. The stimulation of P-450 reductase by Mg++ could add to the stimulation by the type I substrate benzphetamine, and the inhibition of P-450 reductase by aniline could be reversed by added Mg++. Hepatic microsomes from 3-methylcholanthrene-treated rats responded poorly to either Mg++ or benzphetamine (as compared with microsomes from control or phenobarbital-treated rats). Additions of high concentrations of KCl (50-333 mM) could stimulate P-450 reductase slightly and also slightly enhanced the ability of benzphetamine to stimulate P-450 reductase. In the presence of lower concentrations of KCl (up to 100 mM), Mg++ could still stimulate P-450 reductase. At higher KCl concentrations (100-300 mM) Mg++ effects on P-450 reductase were markedly reduced or obliterated. Mg++ effects on hepatic microsomal P-450 reductase are best demonstrated in low ionic strength solutions with non-metal-binding buffers such as N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) and pH between 7.4 and 8.
Footnotes
- Received March 11, 1971.
- Accepted June 23, 1971.
- © 1971 by The Williams & Wilkins Company
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