Rat testosterone 7 alpha-hydroxylase. Isolation, sequence, and expression of cDNA and its developmental regulation and induction by 3-methylcholanthrene.

K. Nagata, Tadashi Matsunaga, J. Gillette, H. V. Gelboin, F. J. Gonzalez

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Abstract

A P-450, designated P-450a, with high testosterone 7 alpha-hydroxylase activity was purified from rat liver microsomes. Specific polyclonal antibody against this P-450 was used to screen a lambda gt11 expression cDNA library and a 1687-base pair cDNA was isolated and sequenced. The deduced protein had 492 amino acids, a calculated Mr of 56,016, and it shared 51 and 45% amino acid similarities to P-450e and P-450f, respectively. Regions of similarity were distributed in distinct areas of high and low similarity along the P-450a primary sequence. P-450a cDNA was introduced into yeast cells using the expression vector pAAH5, and the resultant yeast microsomes contained both a protein of identical electrophoretic mobility to that of rat P-450a and testosterone 7 alpha-hydroxylase activity. These results confirm enzyme reconstitution data and antibody inhibition data that P-450a possesses testosterone 7 alpha-hydroxylase activity. The antibody and cDNA probes were used to examine the mechanism of regulation of P-450a by inducers and during development. P-450a and its mRNA were present at low level in newborn rats and increased to maximal level at 1 week of age in both males and females. At age 12 weeks, however, the P-450a level decreased in males but remained elevated in females. Concomitant with the decrease in P-450a in adult males was an increase in level of another immunologically related P-450. In adult male rats, P-450a was induced almost 5-fold by administration of 3-methylcholanthrene and this induction was the result of an increase in its mRNA. These results establish testosterone 7 alpha-hydroxylase as a member of the P-450e gene family that is developmentally regulated, sex-dependent, and markedly inducible by 3-methylcholanthrene.

Original languageEnglish
Pages (from-to)2787-2793
Number of pages7
JournalJournal of Biological Chemistry
Volume262
Issue number6
Publication statusPublished - 1987 Feb 25
Externally publishedYes

Fingerprint

Methylcholanthrene
Rats
Complementary DNA
Yeast
Antibodies
Yeasts
Amino Acids
Electrophoretic mobility
Messenger RNA
Liver Microsomes
Microsomes
Gene Library
Base Pairing
Liver
Proteins
Genes
Cells
rat Cyp2a1 protein
steroid hormone 7-alpha-hydroxylase
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Rat testosterone 7 alpha-hydroxylase. Isolation, sequence, and expression of cDNA and its developmental regulation and induction by 3-methylcholanthrene. / Nagata, K.; Matsunaga, Tadashi; Gillette, J.; Gelboin, H. V.; Gonzalez, F. J.

In: Journal of Biological Chemistry, Vol. 262, No. 6, 25.02.1987, p. 2787-2793.

Research output: Contribution to journalArticle

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abstract = "A P-450, designated P-450a, with high testosterone 7 alpha-hydroxylase activity was purified from rat liver microsomes. Specific polyclonal antibody against this P-450 was used to screen a lambda gt11 expression cDNA library and a 1687-base pair cDNA was isolated and sequenced. The deduced protein had 492 amino acids, a calculated Mr of 56,016, and it shared 51 and 45{\%} amino acid similarities to P-450e and P-450f, respectively. Regions of similarity were distributed in distinct areas of high and low similarity along the P-450a primary sequence. P-450a cDNA was introduced into yeast cells using the expression vector pAAH5, and the resultant yeast microsomes contained both a protein of identical electrophoretic mobility to that of rat P-450a and testosterone 7 alpha-hydroxylase activity. These results confirm enzyme reconstitution data and antibody inhibition data that P-450a possesses testosterone 7 alpha-hydroxylase activity. The antibody and cDNA probes were used to examine the mechanism of regulation of P-450a by inducers and during development. P-450a and its mRNA were present at low level in newborn rats and increased to maximal level at 1 week of age in both males and females. At age 12 weeks, however, the P-450a level decreased in males but remained elevated in females. Concomitant with the decrease in P-450a in adult males was an increase in level of another immunologically related P-450. In adult male rats, P-450a was induced almost 5-fold by administration of 3-methylcholanthrene and this induction was the result of an increase in its mRNA. These results establish testosterone 7 alpha-hydroxylase as a member of the P-450e gene family that is developmentally regulated, sex-dependent, and markedly inducible by 3-methylcholanthrene.",
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