Steroid 5alpha-reductase in cultured human fibroblasts. Biochemical and genetic evidence for two distinct enzyme activities.

R J Moore,J D Wilson

doi:10.1016/s0021-9258(17)33036-3

Abstract

Various properties of the steroid 5alpha-reductase have been examined in cell-free extracts of skin and of fibroblasts cultured from genital and nongenital skin from control subjects and from patients with several forms of male pseudohermaphroditism. When 20alpha-hydroxy-4-[1,2-3H] pregnen-3-one was used as substrate, two 5alpha-reductase activities could be demonstrated in intact skin and cultured fibroblasts. The major activity, previously described for microsomes from human prepuce and extracts of cultured foreskin fibroblasts, is characterized by a narrow pH optimum near 5.5 and is limited to fibroblasts derived from genital skin. A second activity, not limited by the site of biopsy, has been demonstrated over a higher and broader range of pH (from 7 to 9); this enzyme activity is found in both genital and nongenital skin and in fibroblasts cultured from all skin regions. Whereas there is wide variability in the activity at pH 5.5 in genital skin fibroblasts, the activity at pH 7 to 9 is similar in fibroblasts derived from all anatomical sites. The two activities exhibit different kinetics with respect to steroid substrate and are also dissimilar in their subcellular distributions. Other properties, such as coenzyme requirement, steroid substrate specificity, and instability with increasing temperature, appear to be similar.

Highlights

Various properties of the steroid 5n-reductase have been examined in cell-free extracts of skin and of fibroblasts cultured from genital and nongenital skins from control subjects and from patients with several forms of male pseudohermaphroditism
The activity of steroid 5a-reductase’ in fibroblasts grown familial incomplete male pseudohermaphroditism, type 2) in from human skin varies widely depending on the anatomical which enzyme activity is as low in genital as in nongenital skin site from which the skin is obtained
Fibroblasts grown from sites; similar results were found in fibroblasts grown from perineal skin usually various skin sites leading to the hypothesis that the failure of exhibit high activity, whereas fibroblasts grown from non- normal male sexual differentiation in 46,XY individuals with perineal sites invariably have low the abnormality is the result of a deficiency of the specific activities (l-4)

Summary

MATERIALS AND METHODS

Trihydroxy-5ol-pregnane-3,20-dione: EGTA, ethylene glycol bis(P- Cell Culture-The fibroblasts used in these experiments were aminoethyl ether)N,N’-tetraacetic acid; Tricine, N- [tris(hydroxy- established from explants of genital and nongenital skin from normal methyl)methyl]glycine. Haruesting and Cell Fractionation-Each monolayer was rinsed twice with 5.6 ml of 0.15 M NaCl and incubated 1 h at 37O with 5.6 ml of 10 mM Tris/chloride (pH 7.4), containing 0.15 M NaCI, 1 mM. The particulate fractions were suspended in 10 mM Tris/chloride (pH 7.4) by Dounce homogenization and contained 1 to 2 mg of protein/ml. A cell-free extract was prepared by centrifugation at 450 x g for 10 min and used directly for enzyme assays. At the end of the incubation period the reactions were stopped by the addition of 5 volumes of chloroform/methanol; the steroids were extracted, and aliquots containing approximately. For assays utilizing [1,2m3H]testosterone or [1,2m3H]androstenedione, the nonradioactive carrier steroids were testosterone, androstenedione, dihydrotestosterone, androstanedione, and androstanediol.

RESULTS

B ZOa-HYDROXY-4-PREGNEN-3-ONE

DISCUSSION