Characterization of rat 5alpha-reductases type 1 and type 2 expressed in Saccharomyces cerevisiae.
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.
Poletti
A
A
Instituto de Endocrinologia, Milano, Italy.
Celotti
F
F
Motta
M
M
Martini
L
L
D000818:Animals; D001483:Base Sequence; D002458:Cell Fractionation; D042944:Cholestenone 5 alpha-Reductase; D003001:Cloning, Molecular; D015870:Gene Expression; D007527:Isoenzymes / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010088:Oxidoreductases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae
D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D010088:Oxidoreductases; D042944:Cholestenone 5 alpha-Reductase