Baculovirus-directed expression of human prostatic steroid 5 alpha-reductase 1 in an active form.
In the human prostate, the enzyme steroid 5 alpha-reductase (h5 alpha R) catalyses the conversion of testosterone into the more potent androgen, dihydrotestosterone. Two distinct cDNAs coding for h 5 alpha R in the human prostate have been previously characterized. Enzyme h5 alpha R1 shows a maximum activity at basic pH whereas h5 alpha R2 has an acidic pH optimum activity. We report here the expression of the human steroid h5 alpha R1 in a eukaryotic expression system: the baculovirus-directed-insect cell expression system. The full length cDNA was inserted into the Autographa californica nuclear polyhedrosis virus genome and expressed in Spodoptera frugiperda, Sf9, insect cells. Sf9 cells were infected with the recombinant baculovirus and homogenates used in h5 alpha R activity assays by high pressure liquid chromatography showed that a catalytically active enzyme was produced. The recombinant enzyme showed an apparent Km for testosterone of 2.07 microM and a V(max) of 10.1 nmol of dihydrotestosterone/ min/mg of protein. Recombinant h 5 alpha R1 activity was inhibited by specific h 5 alpha R inhibitors such as 4-MA (Ki = 2.6 nM). Subcellular distribution in Sf9 cells demonstrated that the enzyme was associated with the nuclear membrane.
Iehlé
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C
Laboratoire de Cancérologie Expérimentale, Faculté de Médecine, Marseille, France.
Délos
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Filhol
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Martin
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PM
D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology; D003001:Cloning, Molecular; D006801:Humans; D008297:Male; D008861:Microsomes / Q000201:enzymology; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D011467:Prostate / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera
D011994:Recombinant Proteins; D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase