Abstract
5 alpha-Androstane-3 beta, 17 beta-diol hydroxylase (3 beta-diol hydroxylase), a form of cytochrome P-450, was purified from rat ventral prostate, and its regulation as a function of age and 5 alpha-dihydrotestosterone (DHT) treatment was examined. Cytochrome P-450 could be quantitated by its CO difference spectrum only after partial purification from the microsomal membrane, and this was achieved by chromatography on p-chloroamphetamine-coupled Sepharose. Further purification of prostate microsomal P-450 by anion exchange chromatography yielded a preparation with a P-450 content of 8-10 nmol/mg of protein, which upon sodium dodecyl sulfate electrophoresis showed, in the molecular weight region between 50,000 and 60,000 where P-450 is expected to migrate, a single protein band of Mr 54,000. This preparation upon reconstitution with cytochrome P-450 reductase and microsomal lipid catalyzed the formation of three triols, 5 alpha-androstane-3 beta, 7 beta, 17 beta-triol, 5 alpha-androstane-3 beta, 6 alpha, 17 beta-triol, and 5 alpha-androstane-3 beta, 7 alpha, 17 beta-triol from 3 beta-diol in the ratio 1:7:3. Both turnover number and the ratio of the three products in the reconstituted system were similar to that found in prostate microsomes. These data indicate that a single form of P-450 catalyzes the formation of all three triols and that 3 beta-diol hydroxylase is the major, if not the only, form of P-450 in the prostate microsomes of untreated rats. The yield of P-450 from prostate microsomes varied as a function of age from a high level of 0.05 nmol/mg of microsomal protein in 6-week-old rats to 0.002 nmol/mg of microsomal protein in rats 11 weeks or older. 3 beta-Diol hydroxylase activity followed a similar age-related pattern varying between 2,000 and 4,000 nmol of triols formed/g of tissue/h in 6-week-old rats to 100 nmol of triols formed/g of tissue/h in 11-week-old rats. Treatment of 6-week-old rats with DHT did not prevent the age-related decrease in 3 beta-diol hydroxylase activity. However, DHT does play a role in the regulation of this enzyme since castration resulted in a loss of catalytic activity from the prostate and treatment of castrated rats with DHT caused an induction of the enzyme.
Highlights
5a-Androstane-3t9,17B-diolhydroxylase (38-diolhy- hydroxysteroid oxidoreductase to 5a-androstane-3a,17j3-diol droxylase), a form of cytochrome P-450, was purifiedand 5a-androstane-3P,l7/3-dio(l3@-diol),respecfrom rat ventral prostate, and its regulation as a func- tively
Cytochrome P-450 could be quantitated by its CO found in normal and hyperplastic human and canine prostate differencespectrum only afterpartial purification [5, 6] as well as in normal rat prostate [3, 4] and other DHT
Preparation upon reconstitution with cytochrome P- The enzymes catalyzing the 6a- and 7a-hydroxylations of 450 reductase andmicrosomal lipid catalyzed the for- 3P-diol have been characterized kinetically and by their submation of three triols, 5a-androstane-3&78,17&triol, strate specificity in rat prostate microsomes [3, 4] and have
Summary
Dept. of Pharmacology, McGill University, and thetwo ventral prostate lobes were excised, dissected free of fat, Montreal H3G 1Y6, Canada. After a 5000 X g centrifugation for ication (3 X 30 s) in 50 mM potassium phosphate buffer, pH 7.4, at a 10 min (Beckman 52-21 centrifuge, rotor JA-17 or JA-20),the pellet concentration of 1mg/ml. The sample A 5000 liquid chromatograph HPLC pump (Varian AB, Solna, Swewas left for 30 min a t 4 "C and was diluted to one-fourth with 5 den) equipped with a C18-column (46 mm X 25 cm) (Beckman) was mM sodium phosphate buffer, pH 7.5, containing 20% glycerol, giving used. The samples were taken to dryness with nitrogen, redissolved the same concentration of sodium cholate and Emulgen 911 as in the in 30 p1 of methano1:water (4555, v/v), and 20 pl of this volume was starting buffer. Cytochrome P-450 was eluted 3/3,6a,l7~-triol and5a-androstane-3fl,7a,17P-triowlere identified by with solubilization buffer. 2 mg of hemoglobin (Sigma H-2500) in 2 ml of solubilization buffer et al [4]
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