Purification and properties of steroid 17 alpha-hydroxylase from calf testis.
Steroid 17 alpha-hydroxylase has emerged as a key enzyme in steroidogenic cells: (i) it represents the branch point between the 17-deoxy (mineralo) and the 17-hydroxy (gluco) corticosteroid pathways in the adrenal cortex; (ii) the corresponding specific cytochrome (P-450(17 alpha] is highly dependent upon hormonal regulation; and (iii) the enzyme also catalyzes the steroid 17-20 lyase reaction, leading to the major androgens in the testis. As a prerequisite to the study of its regulation in intact cell, 17 alpha-hydroxylase was purified from calf testis microsomal preparations. Following five chromatographic steps, the enzyme was obtained as an apparently homogeneous protein of Mr = 57 kDa upon gel electrophoresis. The procedure yielded a recovery of about 10% as judged by cytochrome P-450 assay. Whereas 17 alpha-hydroxylase specific activity was about 30-fold enriched during the purification, that of the C17-20 lyase was increased by about 6-fold, strongly suggesting that its organelle environment may modulate the enzymatic activity. The purified enzyme yielded a 20 N-terminal amino-acid sequence showing a complete homology with that of its adrenal counterpart and a polyclonal antibody raised against our preparation revealed a 57 kDa protein band in bovine adrenocortical microsomal extracts, upon immunoblotting experiments. It was thus concluded that bovine 17 alpha-hydroxylase activity is supported by highly similar if not identical enzymatic proteins in both testis and adrenal cortex tissues. The purified P-450(17 alpha) preparation is now being used in reconstitution experiments which suggest that microsomal components may contribute to a different expression of the enzyme specificity in its native testis or adrenocortical intracellular environment, respectively.
Perrin
A
A
INSERM U 244 Centre d'Etudes Nucléaires, Grenoble, France.
Defaye
G
G
Guidicelli
C
C
Chambaz
E M
EM
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D015153:Blotting, Western; D002384:Catalysis; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D005779:Immunodiffusion; D008297:Male; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification / Q000378:metabolism; D013254:Steroid 17-alpha-Hydroxylase / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine; D013737:Testis / Q000201:enzymology*
D000596:Amino Acids; D013254:Steroid 17-alpha-Hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase