Studies on cytochrome P-450 (P-450 17 alpha,lyase) from guinea pig adrenal microsomes. Dual function of a single enzyme and effect of cytochrome b5.
We have reported (Kominami, S., Shinzawa K. and Takemori, S. (1982) Biochem. Biophys. Res. Commun. 109, 916-921) that a cytochrome P-450 purified from guinea pig adrenal microsomes shows 17 alpha-hydroxylase and C-17,20-lyase activities in a reconstituted system with NADPH-cytochrome P-450 reductase. The homogeneity of the purified cytochrome P-450 was examined with the following methods: isoelectric focusing, immunoelectrophoresis and affinity chromatography on cytochrome b5-immobilized Sepharose. It was found that progesterone competitively inhibited C-17,20-lyase reaction and that progesterone was converted into androstenedione by 17 alpha-hydroxylation followed by the lyase reaction. These results indicate that the dual activities are carried out by a single enzyme (P-450 17 alpha,lyase). P-450 17 alpha,lyase had the maximum activity at pH 6.1 both for 17 alpha-hydroxylation (6.0 nmol/min per nmol of P-450) and the lyase reaction (11.0 nmol/min per nmol of P-450). Upon addition of cytochrome b5 to the reconstituted system, the optimal pH for 17 alpha-hydroxylation was shifted to 7.0 and that of the lyase reaction to 6.6. The maximum activities at these optimal pH values were almost the same in the presence or absence of cytochrome b5. With the addition of cytochrome b5, both the activities were stimulated above pH 6.3-6.5 and were suppressed below pH 6.3-6.5. These results indicate that cytochrome b5 plays some important role in controlling the dual activities of P-450 17alpha,lyase.
D000311:Adrenal Glands / Q000201:enzymology*; D000446:Aldehyde-Lyases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D000818:Animals; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification* / Q000502:physiology; D003573:Cytochrome b Group / Q000302:isolation & purification* / Q000502:physiology; D015786:Cytochromes b5; D004789:Enzyme Activation; D006168:Guinea Pigs; D006863:Hydrogen-Ion Concentration; D008861:Microsomes / Q000201:enzymology*; D013254:Steroid 17-alpha-Hydroxylase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D013250:Steroid Hydroxylases / Q000302:isolation & purification*
D003573:Cytochrome b Group; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D013254:Steroid 17-alpha-Hydroxylase; D000446:Aldehyde-Lyases