Solubilization of human prostatic 5 alpha-reductase.
A sensitive assay for 5 alpha-reductase was introduced which is capable of detecting at least 0.2 U of activity per sample. The assay was used in developing a method for the solubilization of human prostatic 5 alpha-reductase. Homogenisation conditions were devised under which 95% of the total prostatic 5 alpha-reductase was released into the microsomal fraction. A combination of 0.1 M sodium citrate, 0.1 M KCl, 20% (v/v) glycerol, 0.5 mM NADPH and 1 microM testosterone was found to stabilise 5 alpha-reductase in the presence of detergents. The effect of the presence of low concentrations of detergents in the assay on the activity of 5 alpha-reductase was studied. Triton X-100, Lubrol PX and Nonidet P-40, caused a concentration-dependent inhibition of activity. The ability of several detergents (Triton X-100 MEGA-9, Tween 20, Tween 80, digitonin, Lubrol PX and Nonidet P-40) to solubilise 5 alpha-reductase was studied. All detergents caused a concentration-dependent solubilization of 5 alpha-reductase. Significant amounts of active solubilized enzyme were recovered only with Lubrol PX at concentrations less than 1.1 mg/ml. Seventy percent of the 5 alpha-reductase was solubilized in an active form by extracting the membranes 3 times with 0.8 mg/ml Lubrol PX.
D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase / Q000302:isolation & purification*; D003902:Detergents; D006801:Humans; D008297:Male; D008722:Methods; D010088:Oxidoreductases / Q000302:isolation & purification*; D000077423:Polidocanol; D011092:Polyethylene Glycols; D011467:Prostate / Q000201:enzymology*; D012995:Solubility; D013501:Surface-Active Agents
D003902:Detergents; D013501:Surface-Active Agents; D000077423:Polidocanol; D011092:Polyethylene Glycols; D010088:Oxidoreductases; D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase