Properties of 4-ene-5 alpha-reductase and studies on its solubilization from porcine testicular microsomes.
The activity of 4-ene-5 alpha-reductase was assayed in porcine testis homogenates and subcellular fractions, using testosterone as substrate. 'Marker' enzyme activities were utilized to indicate the purity of the subcellular fractions. 4-Ene-5 alpha-reductase activity was associated with the microsomal fraction; there was no activity in the purified nuclear fraction. Enzyme activity was higher in the testes of 6 week old pigs than those of 3 and 17 week old animals, and a range of activity was found. The enzyme was unstable when stored at -20 degrees C but the addition of albumin (0.1%, w/v) or glycerol (20%, v/v) to the buffer and storage at -70 degrees C or in liquid nitrogen ensured that maximal activity was retained for at least 35 days. In addition to 5 alpha-DHT, other 5 alpha-reduced metabolites and 4-androstenedione were formed in this reaction; NADPH was the preferred cofactor, but 40% of the 4-ene-5 alpha-reductase activity was retained when NADH was used. Solubilization of the microsomal enzyme was achieved using sodium citrate (0.1 M); 4-ene-5 alpha-reductase activity was enhanced to greater than 120% and 60% of this activity was in the soluble fraction. The optimum pH and temperature for both soluble and membrane-bound 4-ene-5 alpha-reductase were 6.9 and 32 degrees C, respectively. The mean apparent Km and Vmax were 0.6 mumol/l and 158 pmol/min/mg microsomal protein for the microsomal enzyme and 1.42 mumol/l and 212.0 pmol/min/mg soluble protein for the solubilized 4-ene-5 alpha-reductase. The estimated sedimentation coefficient was 11.6.
Watkins
W J
WJ
Division of Biochemistry, United Medical School (Guy's Hospital), London, England.
Goldring
C E
CE
Gower
D B
DB
D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology; D042944:Cholestenone 5 alpha-Reductase; D003600:Cytosol / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D008861:Microsomes / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology; D009249:NADP / Q000494:pharmacology; D010088:Oxidoreductases / Q000378:metabolism*; D012995:Solubility; D013552:Swine; D013696:Temperature; D013737:Testis / Q000201:enzymology* / Q000254:growth & development; D013739:Testosterone / Q000378:metabolism
D013739:Testosterone; D009249:NADP; D010088:Oxidoreductases; D042944:Cholestenone 5 alpha-Reductase