Thigpen A E, Cala K M, Russell D W
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75235.
J Biol Chem. 1993 Aug 15;268(23):17404-12.
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.
类固醇5α-还原酶的膜结合同工酶，分别命名为1型和2型，可合成强效雄激素双氢睾酮。1型同工酶的最适pH值为碱性（7.0 - 8.5），而2型同工酶的最适pH值为酸性（5.0）。为深入了解这一神秘差异，建立了表达人5α-还原酶同工酶的中国仓鼠卵巢细胞系。两种蛋白质的半衰期均大于30小时，且不受4-氮杂甾体抑制剂非那雄胺和17β-（N，N - 二乙基）氨基甲酰基-4-甲基-4-氮杂-5α-雄甾烷-3-酮的影响。纳摩尔浓度的非那雄胺可阻断抗肽抗体对2型同工酶的免疫沉淀，这表明药物结合会改变蛋白质构象。相比之下，非那雄胺（50μM）对1型同工酶的免疫沉淀没有影响。通过免疫细胞化学方法发现，两种同工酶均定位于内质网，且其羧基末端暴露于细胞质中。在细胞裂解物中，2型同工酶在pH 5.0时表现出最大反应速度（Vmax），但在中性pH下具有更高的底物亲和力。在完整细胞和通透细胞中，2型同工酶的表观底物米氏常数（Km）与在中性pH下细胞裂解物中测定的结果相似。结果表明，2型同工酶在中性pH下效率更高，而在裂解物中测定的酸性最适pH值是细胞裂解的结果。
