Properties of 4-ene-5α-reductase and studies on its solubilization from porcine testicular microsomes

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Abstract

The activity of 4-ene-5α-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-5α -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°C but the addition of albumin (0.1%, w/v) or glycerol (20%, v/v) to the buffer and storage at − 70°C or in liquid nitrogen ensured that maximal activity was retained for at least 35 days. In addition to 5α-DHT, other 5α -reduced metabolites and 4-androstenedione were formed in this reaction; NADPH was the preferred cofactor, but 40% of the 4-ene-5α-reductase activity was retained when NADH was used. Solubilization of the microsomal enzyme was achieved using sodium citrate (0.1 M); 4-ene-5α-reductase activity was enhanced to >120% and 60% of this activity was in the soluble fraction. The optimum pH and temperature for both soluble and membrane-bound 4-ene-5α-reductase were 6.9 and 32°C, respectively. The mean apparent Km and Vmax were 0.6 μmol/1 and 158pmol/min/mg microsomal protein for the microsomal enzyme and 1.42μmol/l and 212.0 pmol/min/mg soluble protein for the solubilized 4-ene-5α-reductase. The estimated sedimentation coefficient was 11.6.

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