Biochemical characterization of the NADPH: 4-ene-3-ketosteroid 5α-oxidoreductase in rat ovarian suspension cultures
| Autor: | P. De Moor, Ph. Koninckx, Guido Verhoeven, Walter Heyns |
|---|---|
| Rok vydání: | 1979 |
| Předmět: |
Biochemistry
Cofactor chemistry.chemical_compound Endocrinology 3-Oxo-5-alpha-Steroid 4-Dehydrogenase Suspensions Oxidoreductase Ketosteroid Animals Cells Cultured chemistry.chemical_classification biology Ovary Substrate (chemistry) Epitestosterone Testosterone 5 alpha reductase Aminoglutethimide Enzyme assay Rats Enzyme chemistry biology.protein Female Oxidoreductases Uncompetitive inhibitor |
| Zdroj: | Journal of Steroid Biochemistry. 10:325-333 |
| ISSN: | 0022-4731 |
| DOI: | 10.1016/0022-4731(79)90260-7 |
| Popis: | A method was devised to measure the NADPH:4-ene-3-ketosteroid 5α-oxidoreductase of ovarian cells, in suspension or in monolayer culture. The enzymatic activity was assayed by measuring initial conversion rates of [ 3 H]-17-epitestosterone to its 5α-reduced metabolites. With this substrate no 17-keto derivatives were formed, thus facilitating the Chromatographic separation of the reaction products. Before and during the incubation with the tritiated substrate, aminoglutethimide (10 −4 M) was added to the incubation medium in order to block the endogeneous steroid production. The characteristics of the 5α-reductase in living cells were investigated and compared with those found in ovarian homogenates. In living cells the apparent K M approximated 0.78 μM. The 5α-reductase of 17-epitestosterone was inhibited by 4-ene-3-keto-steroids without an oxygen substitution on carbon 11 but not by 5α-reduced steroids. 17β-Estradiol in high concentration functions as an uncompetitive inhibitor. In homogenates an apparent K M of 0.57 μM was measured. The enzyme exhibited a pH optimum around 7 and required NADPH as cofactor. Inhibition of the enzyme activity by 17β-estradiol could not be demonstrated under these conditions. The characteristics of the 5α-reductase in the rat ovary are thus comparable to those described in androgen target cells. The activity of the 5α-reductase in living cells was comparable with the activity found in homogenates supplemented with NADPH. Cofactor availability is thus not a limiting factor for 5α-reductase activity in ovarian cells. |
| Databáze: | OpenAIRE |
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