Isolation by ion-exchange high performance liquid chromatography of rabbit liver cytochrome P-450 with regioselectivity for omega-hydroxylation of prostaglandins.
Previous studies suggested that rabbit liver microsomes contain cytochrome P-450 monooxygenase(s) with low affinity for (omega-1)-hydroxylation and high affinity for omega-hydroxylation of prostaglandins (Theoharides, A. D., and Kupfer, D. (1981) J. Biol. Chem. 256, 2168-2175). The current investigation describes the isolation from livers of untreated rabbits of a cytochrome P-450 catalyzing, with regioselectivity, the omega-hydroxylation of prostaglandins E1 and E2. The isolation of the enzyme involved enrichment of the omega-hydroxylase activity by polyethylene glycol 8000 fractionation, followed by ion-exchange high performance liquid chromatography. Based on Mr of 59,000-60,000 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the isolated enzyme is referred to as P-450 form 7. This P-450 exhibits a low spin spectrum (lambda max = 417 nm) and a difference spectrum of the CO-reduced complex versus reduced (lambda max = 451 nm). For catalytic activity, the P-450 form 7 was reconstituted with NADPH-P-450 reductase, cytochrome b5, and lipid. There was no activity in the absence of the reductase, and deletion of cytochrome b5 yielded a minimal amount of product (heme could not substitute for cytochrome b5), demonstrating an absolute requirement for these components.
D000527:Alprostadil; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification* / Q000378:metabolism; D003573:Cytochrome b Group / Q000494:pharmacology; D015786:Cytochromes b5; D015232:Dinoprostone; D004591:Electrophoresis, Polyacrylamide Gel; D006900:Hydroxylation; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D009251:NADPH-Ferrihemoprotein Reductase / Q000494:pharmacology; D011458:Prostaglandins E / Q000378:metabolism*; D011817:Rabbits; D013379:Substrate Specificity
D003573:Cytochrome b Group; D011458:Prostaglandins E; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; D000527:Alprostadil; D015232:Dinoprostone