Isolation by ion-exchange high performance liquid chromatography of rabbit liver cytochrome P-450 with regioselectivity for omega-hydroxylation of prostaglandins

• Published in: The Journal of biological chemistry Vol. 260; no. 4; pp. 2027 - 2030
• Main Authors: Holm, K A, Kupfer, D
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 25.02.1985; American Society for Biochemistry and Molecular Biology
• Subjects: Alprostadil; Animals; Chromatography, High Pressure Liquid; Cytochrome b Group - pharmacology; Cytochrome P-450 Enzyme System - isolation & purification; Cytochrome P-450 Enzyme System - metabolism; Cytochromes b5; Dinoprostone; Electrophoresis, Polyacrylamide Gel; Hydroxylation; Male; Microsomes, Liver - enzymology; Molecular Weight; NADPH-Ferrihemoprotein Reductase - pharmacology; Prostaglandins E - metabolism; Rabbits; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)89509-6

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.