Microsomal cytochrome P-450 from neonatal pig testis. Purification and properties of A C21 steroid side-chain cleavage system (17 alpha-hydroxylase-C17,20 lyase).
A cytochrome P-450 from neonatal pig testicular microsomes was purified to homogeneity as judged by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels and by double diffusion on agar against antiserum raised in rabbits against the protein. The enzyme shows both 17 alpha-hydroxylase (Vmax = 4.6 nmol of product/min/nmol of P-450, Km = 1.5 microM) and C17,20 lyase (Vmax = 2.6 nmol of product/min/nmol of P-450, Km = 2.4 microM) activities. Both activities require NADPH and a flavoprotein P-450 reductase; microsomal P-450 reductase from pig and rat livers was used in these studies. The enzyme possesses a single subunit of molecular weight 59,000 +/- 1,000 as determined by electrophoresis on polyacrylamide with sodium dodecyl sulfate and by chromatography on sodium dodecyl sulfate-Sephadex. The enzyme is a glycoprotein and contains 8 nmol of heme/mg of protein and 40 nmol of phospholipid/mg of protein. All heme detected by pyridine hemochromogen is accounted for as P-450 by difference spectroscopy of the reduced P-450.carbon monoxide complex. This complex shows an absorbance maximum at 448 nm with no evidence of P-420. These studies raise the possibility that one microsomal protein (cytochrome P-450) may possess two enzymatic activities (hydroxylase and lyase).
D000446:Aldehyde-Lyases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D000831:Animals, Newborn; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D006418:Heme / Q000032:analysis; D006908:Hydroxyprogesterones / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008861:Microsomes / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D013254:Steroid 17-alpha-Hydroxylase / Q000302:isolation & purification / Q000378:metabolism*; D013250:Steroid Hydroxylases / Q000378:metabolism*; D013552:Swine; D013737:Testis / Q000201:enzymology*
D006908:Hydroxyprogesterones; D009097:Multienzyme Complexes; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D013254:Steroid 17-alpha-Hydroxylase; D000446:Aldehyde-Lyases