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Arch Biochem Biophys. 1987 Oct;258(1):292-7.

Purification and characterization of ethanol-inducible human hepatic cytochrome P-450HLj.

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1
Division of Clinical Toxicology and Environmental Medicine, Medical College of Virginia, Richmond 23298.

Abstract

Human hepatic cytochrome P-450HLj was purified in a catalytically active state from microsomes obtained from an ethanol-intoxicated man. The electrophoretically homogeneous preparation of HLj was compared to rat P-450j and found to have a slightly different apparent molecular mass (54 vs 51.5 kDa) but highly similar immunochemical, spectral, and catalytic properties. Purified HLj exhibited high activity toward N-nitro-sodimethylamine (NDMA) and aniline metabolism, low but measurable activity toward benzphetamine and 7-ethoxycoumarin, and no detectable activity toward benzo[a]pyrene, testosterone, and progesterone. Antibody against rat P-450j reacted with HLj in immunoblot analyses and, when added directly to HLj before reconstitution with NADPH-cytochrome P-450 reductase and lipid, the antibody inhibited (96%) NDMA metabolism by HLj almost completely. However, if HLj was reconstituted with the other components before the addition of the anti-P-450j IgG, the ability of the antibody to inhibit the metabolism of NDMA was greatly diminished. This suggests that the interactions between reductase and HLj are similar to those previously observed between rat P-450j and reductase, and appear to prevent the complete access of anti-P-450j. The addition of cytochrome b5 to reconstitution systems containing HLj resulted in a small increase in the Vmax from NDMA demethylation accompanied by a decrease in Km,app (1.3 to 0.3 mM) as has been observed in reconstitution systems with rat P-450j. Therefore, in reconstituted systems, cytochrome b5 appears to play an important role in the biotransformations mediated by HLj and P-450j. In conclusion, this study demonstrates that HLj is functionally related to ethanol-inducible rat P-450j and rabbit LM3a.

PMID:
3310902
[Indexed for MEDLINE]
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