Characterization of Chinese hamster ovary cell lines expressing human steroid 5α-reductase isozymes

Anice E. Thigpen, Kristine M. Cala, David W. Russell

Research output: Contribution to journalArticlepeer-review

137 Scopus citations

Abstract

Membrane-bound isozymes of steroid 5α-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 5α-reductase isozymes were established. The half-lives of both proteins are >30 h and are not altered by the 4-azasteroid inhibitors finasteride and 17β-(N, N,-diethyl)carbamoyl-4- methyl-4-aza-5α-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 μM) 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 V(max) at pH 5.0 but has a higher substrate affinity at neutral pH. In intact and permeabilized cells, isozyme 2 has an apparent substrate K(m) 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.

Original languageEnglish (US)
Pages (from-to)17404-17412
Number of pages9
JournalJournal of Biological Chemistry
Volume268
Issue number23
StatePublished - 1993

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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