P-450 human-2 is a human cytochrome P-450 that is immunochemically related to a constitutive male-specific cytochrome P-450 (P-450-male) and the phenobarbital-inducible P-450b/e in rat liver. By screening a human liver cDNA library in bacteriophage lambda gt11, we isolated a clone with an insert length of 1,847 bases (pHY13). The clone was sequenced and shown to code for a protein of 487 amino acids. The N-terminal 11-amino-acid sequence was in agreement with the protein sequence of P-450 human-2. The nucleotide sequence of pHY13 showed less than 50% similarity with those of human cytochrome P-450s, pHP-450(1), HLp, P-450NF, P1-450 4, and P3(450), but the nucleotide sequence of pHY13 is 80% similar to the reported sequence of rat cytochrome P-450, P-450(M-1). In addition, the coding sequence of pHY13 showed close similarity to that of MP-8, which was recently reported as the sequence corresponding to human cytochrome P-450MP, although no apparent similarity was observed in their 3' non-coding sequences except for the first 75 bases and the expected length of the complete sequences. These results, together with the immunochemical data, indicate that P-450 human-2 is closely related, but not identical, to P-450MP, and may belong to the category of developmentally regulated constitutive cytochrome P-450s.
Two forms of cytochrome P-450 (P-450 human-1 and P-450 human-2) have been purified from human liver microsomes to electrophoretic homogeneity. P-450 human-1 and P-450 human-2 differ in their apparent molecular weights (52,000 and 56,000, respectively) and Soret peak maxima in the CO-binding reduced difference spectrum (447.6 and 450.3 nm, respectively). In the reconstituted system using rat liver NADPH-cytochrome c (P-450) reductase, P-450 human-2 more effectively oxidized benzo(a)pyrene (80-fold), ethylmorphine (2-fold), and 7-ethoxycoumarin (2-fold) than did P-450 human-1. However, P-450 human-1 showed higher testosterone 6 beta-hydroxylase activity, and the activity was markedly increased by the inclusion of cytochrome b5 or spermine in the reconstituted system. Antibodies raised against P-450 human-1 inhibited more than 80% of microsomal testosterone 6 beta-hydroxylase activity in human liver. Immunoblotting analysis using anti-P-450 human-1 IgG revealed a single immuno-staining band near Mr 52,000 in all human liver samples examined. The amount of immunochemically determined P-450 human-1 varied in parallel with the testosterone 6 beta-hydroxylase activity in human liver. These results indicate that P-450 human-1 is a major form of cytochrome P-450 responsible for microsomal testosterone 6 beta-hydroxylation. Thus, this paper is the first report on human cytochrome P-450 responsible for testosterone 6 beta-hydroxylation, which is the major hydroxylation pathway in human liver microsomes.
The relationship between growth hormone and testosterone (T) 6 beta-hydroxylase was investigated in normal and hypophysectomized (hypox) male rats. The administration of human growth hormone (hGH) by either intermittent injections or continuous infusion to normal and hypox rats decreased the activity of T 6 beta-hydroxylase in hepatic microsomes. Hypophysectomy did not reduce, but rather increased the 6 beta-hydroxylase activity, while the 16 alpha-hydroxylase activity decreased. These results, together with the data from a Western blot, indicate that growth hormone acts as a repressive factor for the expression of T 6 beta-hydroxylase in a manner different from the regulation of T 16 alpha-hydroxylase.
Abstract-Wehave examined the effect of dietary protein deficiency on rat hepatic drug-metabolizing enzyme system for a period of two months. Cytochrome P-450 and b5 contents in liver microsomes, which were plotted on semilogarithmic paper as a function of the time of deficiency, showed biphasical reductions during protein deficiency: rapid decreases in the first 3 weeks were followed by more gradual decreases. However, the three enzymatic activities examined, i.e. aminopyrine demethylase, aniline hydroxylase and p-nitroanisole demethylase, were not reduced at a uniform rate. In the earlier phase, activities of the former two enzymes were reduced more rapidly than that of the last phase. This biphasical and non-uniform reduction of enzymatic activities suggests the existence of two or more cytochrome P-450 subspecies in nondepleted male rats. Intraperitoneal administration of well-known environmental pollutants, polychlorinated dibenzofurans and biphenyls (100 fig and 100 mg/kg, respectively) to the depleted rats resulted in a marked induction of drug-metabolizing enzymes. However, as the deficiency became more severe (2 months), the induction declined to a considerable degree, especially in the case of polychlorinated biphenyl administration.In 1952, V.A. Drill presented a review concerning the interaction between hepatotoxins and dietary factors, in which he showed that protein deficiency affected the toxicity of the various hepatotoxins (1). Since then, many laboratories have shown that protein deficiency decreases the microsomal oxidation rates of a great variety of drugs and other foreign chemicals (2-4). Boyd and his colleagues (5, 6) observed that many pesticides indicated increased toxicity in protein-depleted animals, as illustrated by a 2,100-fold increase in the toxicity of captan (5). In contrast, some compounds such as carbon tetrachloride (7) and heptachlor (8) have shown lower toxicities. These increased or decreased toxicities observed in protein-depleted animals are generally interpreted to be caused by the depression of foreign chemical metabolism during protein deficiency.In the last decade, several investigators suggested that different forms of cytochrome P-450 existed in liver microsomes, and their arguments were actually proved by the purification of several cy tochrome P-450 and P-448 subspecies different in spectral properties, minimal molecular weight, substrate specificities or immunological properties (9).In the present report we have examined the time course of the decrease of rat hepatic hemoprotein contents and drug-oxidation activities during protein deficiency, and obtained results suggesting that different cytochrome P-450 subspecies were reduced at very different rates. We have also examined the induction of these enzymes caused by polychlorinated
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