ASCORBIC-ACID AND CYTOCHROME-P-450

Abstract

Liver microsomal cytochrome P-450 is significantly reduced in ascorbic acid-deficient guinea pigs, and the biochemical basis for this effect was studied. The activities of the key enzymes involved in heme synthesis, .delta.-aminolevulinic acid (ALA) synthetase, ALA dehydratase and ferrochelatase, were not significantly reduced in livers from ascorbic acid-deficient animals. There was no significant difference in the amount of mitochondrial heme in normal and ascorbic acid-deficient livers. Ascorbic acid deficiency did affect induction with diethyl-1,4-dihydro-2,4,6-trimethylpyridine-3,5-dicarboxylate; a 6-fold increase in ALA synthetase activity occurred in liver homogenates prepared from normal animals in contrast to no significant increase in homogenates prepared from ascorbic acid-deficient animals. Multiple forms of cytochrome P-450 exist in guinea pig microsomes as demonstrated in microsomes from other species. Separation of 44,000-60,000 dalton polypeptides (MW region for the various forms of cytochrome P-450) by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed quantitative differences in the polypeptides from normal and ascorbic acid-deficient microsomes. Ascorbic acid-deficient microsomes consistently demonstrated reductions in 3 polypeptide bands (MW 44,000, 52,000 and 57,000) and increases in 2 polypeptide bands (54,000 and 55,000) compared with normal microsomes. Evidence that these polypeptides are cytochrome P-450 was obtained from heme staining with tetramethylbenzidine and from induction studies with phenobarbital and 3-methylcholanthrene. Apparently ascorbic acid deficiency does not affect the availability of heme for cytochrome P-450 synthesis, and the effect of ascorbic acid may be on the apoprotein moiety of cytochrome P-450.