Abstract
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.
Highlights
Membrane-boundisozymes of steroid 5ar-reductase, The enzyme steroid 5a-reductase(5a-reductase)’ is one designated 1 and 2, synthesize the potent androgen, such therapeutic target that exists in at least two isozymic dihydrotestosterone
In intact and in cultured cells or Xenopus oocytes, type 1 cDNAs from rat permeabilized cells, isozyme 2 has an apparent sub- and man specify an enzyme with a basic pH optimum that is strate K,s,imilar to that determined in cell lysates at sensitive to certainsteroidaland non-steroidal inhibitors neutral pH
The biochemical basis for the unusual acidic pH optimum The establishment of permanent cell lines that express of the type 2isozyme remains unknown; it is known enzymes of therapeutic interest has facilitated the develop- that both the rat and human type 2 isozymes exhibit this ment of drugs that act on the expressed target
Summary
0 1993 by The American Society for Biochemistry and Molecular Biology, Inc. VOl. 268, No 23, Issue of August 15, pp, 17404-17412,1993 Printed in U.S.A. The apparent K,,, values for munoprecipitation of the type 2 isozyme was not affected by steroid substrates as well as for the NADPH cofactor are the pH of the immunoprecipitation buffer or by isolation of similar to those previously determined in transiently trans- microsomes prior to carrying out the precipitation reaction fected cells (Anderson etal., 1991). Finasteridewas unique in its ability to causaeloss of Immunoprecipitation of 5a-Reductase Isozymes-As shown immunoprecipitability in NP-40 cell lysates since the presin Fig. 1, immunoprecipitation of '%labeled proteins from ence of even high concentrations (50 p ~ o)f 4-MA did not CHO-1827 cellswith a rabbit polyclonal antibody against the affect antibody recognition of the type 2 isozyme (lane 9, type 1 isozyme followed by SDS-polyacrylamide gel electro- lower panel).
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