Abstract
7 alpha-Hydroxy-4-cholesten-3-one 12 alpha-monooxygenase was purified from liver microsomes of phenobarbital-treated rabbits. The purification was carried out by solubilization of microsomes by cholate, fractionation with polyethylene glycol, affinity chromatography on cholate-Sepharose 4B column, hydroxylapatite column chromatography, chromatography on DEAE-Sepharose CL-6B column, and a second hydroxylapatite column chromatography. The purified preparation gave a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained 9.0 nmol of cytochrome P-450/mg of protein, which corresponded to 5.3-fold purification from microsomes on the basis of specific heme content. The specific activity of the enzyme expressed as enzyme activity per mg of enzyme protein was increased 315-fold from microsomes. The molecular weight of the enzyme was estimated to be 56,000 from calibrated polyacrylamide gel electrophoresis. The enzyme-pH curve gave a peak at pH 7.0. The Michaelis constant for 7 alpha-hydroxy-4-cholesten-3-one was 27 microM. Absorption spectra of the oxidized form of the enzyme showed a Soret band at 418 nm. 7 alpha-Hydroxy-4-cholesten-3-one 12 alpha-monooxygenase activity was reconstituted from the purified cytochrome P-450, NADPH-cytochrome P-450 reductase, dilauroylglyceryl-3-phosphorylcholine, and NADPH. The purified enzyme was free from steroid 25-hydroxylase activity and that of 26- or 27-hydroxylase but revealed some activity for benzphetamine N-demethylation. The enzyme activity was not inhibited by metapyrone, aminoglutethimide, and KCN, but was seriously inhibited by nonionic detergents such as Emulgen 913. The enzyme was labile under low buffer concentrations but was stabilized at least for 4 weeks under higher buffer concentration such as 300 mM phosphate buffer.
Highlights
From the Departmentof Biochemistry, Hiroshima University Schoolof Dentistry, Kasumi 1-2-3, Minami-ku, Hiroshima 734,Japan and the $Department of Biological Chemistry, Medical School, The Universityof Michigan, Ann Aibor, Michigan48109
Upon reconstitutionwith NADPH-cytochrome P450reductase and cytochrome b5, the P450 enzyme showed a high activity of l2cr-hydroxylation witha turnover number of 36.6 min” at 37 “C. The omission of either cytochrome P450or NADPH-cytochrome P450 reductase bile, 12a-hydroxylation is currently believed to be characteristic of bile acids and alcohols
An- bilized from rat liver microsomes by Bernhardsson et al [3], tibodies prepared from mouse inhibited the l2cr-hy- and the activity was reconstituted from the crude fraction droxylase activity of rabbit liver microsomesabout containing cytochrome P450 and that containing NADPH
Summary
Phosphate buffer containing 0.4% Emulgen 913, and the column was washed with 40 mM potassium phosphate buffer, and elution was carried out with 180 mM potassium phosphate buffer containing the same detergent. Aliquots were injected into an HPLC-hydroxylapatite column (TSK HA-1000, 7.5 X 150 mm) equilibrated with 25 mM potassium phosphate buffer (pH 7.25) containing 0.1% cholate. Proteins were eluted with linear gradients of preparation was submitted to column chromatography on opotassiumphosphate buffer (pH 7.25) (25-135 mM from 0-80 min and 135-400 mM from 80-90 rnin). Following treatmentwithhydroxylapatiteandCMSepharose 4B, HPLC on hydroxylapatite gave several peaks. As shown in with enzyme activity were recbromatographed on anHA-1000 column Fig. 3, HPLC on an anionexchange column, TSK gel DEAEand dialyzed overnight against 20mM Tris-OAc (pH 7.4) con- 5PW, gave the 12a-hydroxylase iannarrow symmetrical peak ' The abbreviations used are: HPLC, high performance liquid.
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