Poletti A, Celotti F, Motta M, Martini L
Instituto de Endocrinologia, Milano, Italy.
Biochem J. 1996 Mar 15;314 ( Pt 3)(Pt 3):1047-52. doi: 10.1042/bj3141047.
Two isoforms of the rat 5alpha-reductase (5alpha-R), the enzyme that converts testosterone into dihydrotestosterone (DHT), and other delta4-3-keto steroids (e.g. progesterone and corticoids) into their 5alpha-reduced metabolites, have been cloned. In this study, a convenient and efficient system was developed to overexpress the two isoenzymes in Saccharomyces cerevisiae by using the ubiquitin-fusion expression system. Two yeast expression vectors have been prepared, YEpR1 and YEpR2, which code for 5alpha-R type 1 and 5alpha-R type 2 respectively; they contain the copper-responsive yeast metallothionein promoter (CUP1) upstream of the ubiquitin coding sequence, and the full-length rat 5alpha-R type 1 or 5alpha-R type 2 cDNAs in frame to the 3' end of the ubiquitin cDNA. The activity of the two isoenzymes produced in yeast was determined in cell lysates at the enzyme pH optima (type 1, pH 7.5; type 2, pH 5.5) and a possible differential intracellular distribution was also evaluated. The kinetic parameters were: type 1, Km 4.6 microM, Vmax.100.6 micrograms/h per mg of protein; type 2, Km 68.6 nM, Vmax. 0.84 micrograms/h per mg of protein. Yeast cell lysates were fractionated by differential centrifugation and the 5alpha-R type 1 activity was maximal in fractions containing nuclei (1000 g and 2500 g), whereas the maximal activity of 5alpha-R type 2 was present in subcellular fractions sedimenting at higher speeds (20000g). The data indicate that yeasts overexpress the two 5alpha-R isoenzymes, maintaining their native biochemical properties, and that the two isoforms are probably differentially localized within the yeast cell.
大鼠5α-还原酶（5α-R）有两种同工型，该酶可将睾酮转化为二氢睾酮（DHT），并将其他Δ4-3-酮类固醇（如孕酮和皮质类固醇）转化为其5α-还原代谢产物，目前已被克隆。在本研究中，通过使用泛素融合表达系统，开发了一种简便高效的系统，用于在酿酒酵母中过表达这两种同工酶。制备了两种酵母表达载体，即YEpR1和YEpR2，它们分别编码1型5α-R和2型5α-R；它们在泛素编码序列上游包含铜响应酵母金属硫蛋白启动子（CUP1），以及与泛素cDNA 3'端读框内的全长大鼠1型5α-R或2型5α-R cDNA。在酶的最适pH值（1型，pH 7.5；2型，pH 5.5）下，测定酵母中产生的两种同工酶在细胞裂解物中的活性，并评估了可能的细胞内差异分布。动力学参数为：1型，Km 4.6微摩尔，Vmax. 100.6微克/小时/毫克蛋白质；2型，Km 68.纳米，Vmax. 0.84微克/小时/毫克蛋白质。通过差速离心对酵母细胞裂解物进行分级分离，1型5α-R活性在含有细胞核的级分（1000g和2500g）中最高，而2型5α-R的最大活性存在于以更高速度沉淀的亚细胞级分（20000g）中。数据表明，酵母过表达这两种5α-R同工酶，保持了它们的天然生化特性，并且这两种同工型可能在酵母细胞内有不同的定位。
