Solubilization of nuclear steroid 5 alpha-reductase from rat ventral prostate.
delta 4-Steroid-5 alpha-reductase (3-oxo-5 alpha-steroid:NADP+ delta 4-oxidoreductase, EC 1.3.1.22), is a membrane-bound enzyme. In the ventral prostate of the rat, its activity is found within the nuclear envelope. Solubilization of this enzyme can only be achieved in the presence of detergents. We studied the inhibitory effect of various detergents on 5 alpha-reductase activity as a function of detergent concentration, of pH, of incubation time, of salt concentration and of additives to the buffer system. Four detergents (Lubrol WX, CHAPS, L-alpha-lysophosphatidylcholine and octyl D-glucopyranoside) were selected for subsequent solubilization studies. The overall recovery of solubilized enzyme activity was about 30% when compared to 100% of 5 alpha-reductase activity found in freshly prepared nuclei. Up to 20-30% of the nuclear proteins were extracted during the solubilization procedure. Among the various treatments tested, a concentration of 3 mg/ml L-alpha-lysophosphatidylcholine per 10 mg/ml of nuclear protein in the presence of 5 mM MgCl2, 0.1 M KCl, 0.1 M sodium citrate and 5 mM NADPH yielded the maximal enzymic activity of 56%, 15% of the nuclear proteins being solubilized in an active and stable form. The activity in these extracts could be kept stable for 2 days at 4 degrees C with a recovery of 75% of enzymic activity. A 3-fold increase of specific 5 alpha-reductase activity was obtained during solubilization under optimal conditions.
Enderle-Schmitt
U
U
Institut für Anatomie und Zellbiologie, Marburg, F.R.G.
Neuhaus
C
C
Aumüller
G
G
D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase / Q000302:isolation & purification*; D058891:5-alpha Reductase Inhibitors; D000818:Animals; D002458:Cell Fractionation; D002498:Centrifugation; D002793:Cholic Acids / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005960:Glucosides / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D008297:Male; D009685:Nuclear Envelope / Q000201:enzymology; D010713:Phosphatidylcholines / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000494:pharmacology; D011467:Prostate / Q000201:enzymology* / Q000648:ultrastructure; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility
D058891:5-alpha Reductase Inhibitors; D002793:Cholic Acids; D003902:Detergents; D005960:Glucosides; D008244:Lysophosphatidylcholines; D010713:Phosphatidylcholines; C017474:Lubrol WX; C018619:octyl-beta-D-glucoside; D011092:Polyethylene Glycols; D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate