20.BETA.-Hydroxysteroid dehydrogenase of neonatal pig testis: Cofactor requirement and stereospecificity of hydrogen transfer from nicotinamide adenine dinucleotide phosphate, reduced form.

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Abstract
The cofactor requirement of purified 20.beta.-hydroxysteroid dehydrogenase from cytosol fraction of neonatal pig testis, in the reduction of 17.alpha.-hydroxyprogesterone was investigated. The enzyme required .beta.-nicotinamide adenine dinucleotide phosphate, reduced form (.beta.-NADPH) as the preferred cofactor, with an apparent Km value of 17 .mu.M. Furthermore, .alpha.-nicotinamide adenine dinucleotide phosphate, reduced form (.alpha.-NADPH), .beta.-3''-NADPH and .beta.-nicotinamide adenine dinucleotide (.beta.-NADH) were also utilized as hydrogen donors in the reduction at relatively high concentration with apparent Km values of 85.2 .mu.M, 179.2 .mu.M and 1.00 mM, respectively. The optimum pH was 5.5 when .beta.-NADPH was used as the cofactor, while it was 6.0 when .beta.-NADH was used. The hydrogen transfer from the .beta.-NADPH to the product, 17.alpha.,20.beta.-dihydroxypregn-4-en-3-one catalyzed by 20.beta.-hydroxysteroid dehydrogenase was stereospecific, and the 4-pro-S-hydrogen of the nicotinamide moiety was transferred to the product.