Baculovirus-directed expression of human prostatic steroid 5α-reductase 1 in an active form
| Autor: | Pierre-Marie Martin, Sylvie Délos, Catherine Iehlé, Odile Filhol |
|---|---|
| Rok vydání: | 1993 |
| Předmět: |
Male
medicine.drug_class viruses Endocrinology Diabetes and Metabolism medicine.medical_treatment Clinical Biochemistry Sf9 Spodoptera Biology Biochemistry Cell Line Steroid law.invention Endocrinology 3-Oxo-5-alpha-Steroid 4-Dehydrogenase law Microsomes Gene expression medicine Animals Humans Cloning Molecular Molecular Biology Testosterone Cell Nucleus chemistry.chemical_classification fungi Prostate Cell Biology Androgen Molecular biology Nucleopolyhedroviruses Recombinant Proteins Enzyme chemistry Dihydrotestosterone Recombinant DNA Molecular Medicine medicine.drug |
| Zdroj: | The Journal of Steroid Biochemistry and Molecular Biology. 46:177-182 |
| ISSN: | 0960-0760 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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