Watkins W J, Goldring C E, Gower D B
Division of Biochemistry, United Medical School (Guy's Hospital), London, England.
J Steroid Biochem. 1988 Mar;29(3):325-31. doi: 10.1016/0022-4731(88)90034-9.
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.
以睾酮为底物，对猪睾丸匀浆和亚细胞组分中的4-烯-5α-还原酶活性进行了测定。利用“标记”酶活性来指示亚细胞组分的纯度。4-烯-5α-还原酶活性与微粒体组分相关；纯化的核组分中无活性。6周龄猪睾丸中的酶活性高于3周龄和17周龄动物睾丸中的酶活性，且存在一定的活性范围。该酶在-20℃储存时不稳定，但在缓冲液中添加白蛋白（0.1%，w/v）或甘油（20%，v/v）并在-70℃或液氮中储存可确保至少35天内保留最大活性。除了5α-双氢睾酮外，该反应还生成了其他5α-还原代谢产物和4-雄烯二酮；NADPH是首选的辅因子，但使用NADH时仍保留了40%的4-烯-5α-还原酶活性。使用柠檬酸钠（0.1M）实现了微粒体酶的溶解；4-烯-5α-还原酶活性提高到大于120%，其中60%的活性存在于可溶部分。可溶性和膜结合的4-烯-5α-还原酶的最适pH和温度分别为6.9和32℃。微粒体酶的平均表观Km和Vmax分别为0.6μmol/L和158pmol/min/mg微粒体蛋白，溶解的4-烯-5α-还原酶的平均表观Km和Vmax分别为1.42μmol/L和212.0pmol/min/mg可溶性蛋白。估计沉降系数为11.6。
