Characterization of Chinese hamster ovary cell lines expressing human steroid 5 alpha-reductase isozymes.
Membrane-bound isozymes of steroid 5 alpha-reductase, designated 1 and 2, synthesize the potent androgen, dihydrotestosterone. Isozyme 1 has an alkaline pH optimum (7.0-8.5), whereas isozyme 2 has an acidic pH optimum (5.0). To gain insight into this enigmatic difference, Chinese hamster ovarian cell lines expressing the human 5 alpha-reductase isozymes were established. The half-lives of both proteins are > 30 h and are not altered by the 4-azasteroid inhibitors finasteride and 17 beta-(N,N,-diethyl)carbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one. Nanomolar concentrations of finasteride block immunoprecipitation of isozyme 2 by antipeptide antibodies, which suggests that drug binding alters protein conformation. In contrast, finasteride (50 microM) has no effect on immunoprecipitation of isozyme 1. Both isozymes are localized to the endoplasmic reticulum by immunocytochemistry and have their carboxyl termini exposed to the cytoplasm. In cell lysates, isozyme 2 exhibits a Vmax at pH 5.0 but has a higher substrate affinity at neutral pH. In intact and permeabilized cells, isozyme 2 has an apparent substrate Km similar to that determined in cell lysates at neutral pH. The results suggest that isozyme 2 is more efficient at neutral pH and that the acidic pH optimum determined in lysates is a consequence of cell lysis.
Thigpen
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Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75235.
Cala
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Russell
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D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D058891:5-alpha Reductase Inhibitors; D000736:Androstenes / Q000494:pharmacology; D000818:Animals; D001378:Azasteroids / Q000494:pharmacology; D016466:CHO Cells; D002463:Cell Membrane Permeability; D006224:Cricetinae; D018120:Finasteride; D005455:Fluorescent Antibody Technique; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics; D007700:Kinetics; D011233:Precipitin Tests; D011487:Protein Conformation; D014162:Transfection
D058891:5-alpha Reductase Inhibitors; D000736:Androstenes; D001378:Azasteroids; D007527:Isoenzymes; D018120:Finasteride; D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase