A simple procedure for solubilizing 3beta-hydroxysteroid dehydrogenase from microsomes of rat adrenals and testis interstitial cells.
A simple procedure is described for solubilizing microsomal 3beta-hydroxysteroid dehydrogenase (3beta-HSD). Microsomes from rat adrenals or from testicular interstitial cells were incubated for 1 or 2 h at 0 C in a buffer containing NaCl followed by overnight storage at -20 C. Maximum solubilization of 3beta-hydroxy-5beta-androstan-17-one-HSD (androstane-3beta-HSD) was obtained by incubating adrenal microsomes with 1 M NaCl and interstitial cell microsomes with 2 M NaCl. Incubation with NaCl for 1 or 2 h resulted in maximum solubilization; incubation with NaCl for 4, 8 or 24 h did not change the amount of enzyme solubilized. From adrenal microsomes incubated with 1 M NaCl, up to 80% (105.7 millimicron/mg microsomes) of the total androstane-3beta-HSD activity was recovered in the supernatant following centrifugation at 130,000 x g for 1 h. The maximum amount of androstane-3beta-HSD solubilized from interstitial cell microsomes was 56% (29.5 millimicron/mg microsomes) at 2 M NaCl. The "solubilized" androstane-3beta-HSD was retarded when chromatographed on a Sephadex G-200 column and it did not pellet out when centrifuged at 130,000 x g for 15 h. KCL appeared to be equally effective in solubilizing androstane-3beta-HSD from microsomes. Other steroid dehydrogenase activities such as pregnanolone-HSD and 3beta-hydroxy-5alpha-androstan-17-one-HSD were also found in the 130,000 x g supernatant.
D015096:3-Hydroxysteroid Dehydrogenases / Q000302:isolation & purification*; D000311:Adrenal Glands / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D000731:Androstanes; D000818:Animals; D002850:Chromatography, Gel; D066298:In Vitro Techniques; D007985:Leydig Cells / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D008297:Male; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology*; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D012965:Sodium Chloride / Q000494:pharmacology; D012995:Solubility; D013010:Sonication; D013379:Substrate Specificity; D013997:Time Factors
D000731:Androstanes; D012965:Sodium Chloride; D011189:Potassium Chloride; D015096:3-Hydroxysteroid Dehydrogenases