NADPH-initiated cytochrome P450-mediated free metal ion-independent oxidative damage of microsomal proteins. Exclusive prevention by ascorbic acid.

C.K Mukhopadhyay,I.B Chatterjee

doi:10.1016/s0021-9258(17)36845-x

Abstract

In this paper we demonstrate that NADPH-initiated oxidative damage of microsomal proteins occurs in the absence of free metal ions and that this protein oxidation is mediated by cytochrome P450 (cyt P450). Oxidized proteins are rapidly degraded by proteases. Ascorbate (AH2) specifically inhibits free metal ion-independent cyt P450-mediated protein oxidation and thereby prevents subsequent proteolytic degradation. Other scavengers of reactive oxygen species including superoxide dismutase, catalase, and glutathione are ineffective. This is in variance with free metal ion-catalyzed protein oxidation, which is accelerated by AH2 and inhibited by catalase. Oxidative damage of proteins has been assessed by the production of carbonyl groups, bityrosine formation, and tryptophan loss. The mechanism of protein oxidation has been studied using a reconstituted system comprised of purified NADPH-cyt P450 reductase, cyt P450, and isolated microsomal proteins as well as model polypeptides, e.g. poly-L-proline and poly-L-lysine. Cyt P450 Fe3+ is reduced by NADPH-cyt P450 reductase to cyt P450 Fe2+, which consumes oxygen in a stoichiometric proportion to produce cyt P450 Fe2+ O2, the resonance form of which is a perferryl moiety, cyt P450 Fe3+.O2-.. It is proposed that cyt P450 Fe3+.O2-. abstracts hydrogen from amino acid side chains leading to the production of carbonyl derivatives. Tentatively, AH2 prevents protein oxidation by interacting with cyt P450 Fe3+.O2-..

Highlights

EXCLUSIVE PREVENTION BY ASCORBIC ACID*(Received for publication, October 25, 1993, and in revised form, January 21, 1994). From the Department of Biochemistry, University College of Science, Calcutta 700 019, Zndia
In this paper we demonstrate that NADPH-initiated cally relevant of these is the NADPWcytochrome P450reductase/cytochromeP450/Fe(III)/02 system
Cyt P450 FeS+ is reduced by NADPH- comprised of purified NADPH-cyt P450 reductaselcyt P450 and cyt P450 reductase to cyt P450 Fe2+, which consumes isolated microsomal proteinass well as poly-L-proline and polyoxygen in a stoichiometric proportion to produce cyt L-lysine, we have studied the mechanismofsoxidative damage

Summary

EXCLUSIVE PREVENTION BY ASCORBIC ACID*

(Received for publication, October 25, 1993, and in revised form, January 21, 1994). From the Department of Biochemistry, University College of Science, Calcutta 700 019, Zndia. In this paper we demonstrate that NADPH-initiated cally relevant of these is the NADPWcytochrome P450 (cyt oxidative damage of microsomal proteins occurs in the P450)reductase/cytochromeP450/Fe(III)/02 system. Incubation System-Incubation mixture using microsomal suspension contained 1mg of microsomal protein in a final volume of 250 pl of 50 nm potassium phosphate buffer,pH7.4.NADPH(0.24 nm or a s indicated) was added to initiate the reaction. For NADPH-initiated protein oxidation in thereconstituted system, 300 pl (final volume) of the incubation mixture contained 0.55 nmol of cyt P450 and 96 units of NADPH-cyt P450 reductaseand 1 mg of protein in 50 m~ potassium phosphate buffer, pH 7.4. Assay of Carbonyl Content-Protein carbonyls were measured by reaction with DNPH as described by Levine et al [12].After incubation with NADPH for required time in the presence of PMSF and EDTA to minimize proteolytic degradation and loss of protein carbonyl, the protein was precipitated with 20% trichloroacetic acid and the carbonyl content was measured in theprecipitate.

RESULTS

Carbonyl formed

DISCUSSION