Two isoforms of the rat 5α-reductase (5α-R), the enzyme that converts testosterone into dihydrotestosterone (DHT), and other ∆4-3-keto steroids (e.g. progesterone and corticoids) into their 5α-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 5α-R type 1 and 5α-R type 2 respectively; they contain the copper-responsive yeast metallothionein promoter (CUP1) upstream of the ubiquitin coding sequence, and the full-length rat 5α-R type 1 or 5α-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 μM, Vmax. 100.6 μg/h per mg of protein; type 2, Km 68.6 nM, Vmax. 0.84 μg/h per mg of protein. Yeast cell lysates were fractionated by differential centrifugation and the 5α-R type 1 activity was maximal in fractions containing nuclei (1000 g and 2500 g), whereas the maximal activity of 5α-R type 2 was present in subcellular fractions sedimenting at higher speeds (20000 g). The data indicate that yeasts overexpress the two 5α-R isoenzymes, maintaining their native biochemical properties, and that the two isoforms are probably differentially localized within the yeast cell.
Characterization of rat 5α-reductases type 1 and type 2 expressed in Saccharomyces cerevisiae
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Angelo POLETTI, Fabio CELOTTI, Marcella MOTTA, Luciano MARTINI; Characterization of rat 5α-reductases type 1 and type 2 expressed in Saccharomyces cerevisiae. Biochem J 15 March 1996; 314 (3): 1047–1052. doi: https://doi.org/10.1042/bj3141047
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