A procedure is described for the purification of the steroid-induced 3α-hydroxysteroid dehydrogenase of Pseudomonas testosteroni. The purified preparations catalyze the nicotinamide-adenine dinucleotide dependent oxidation of about 300,umoles of androsterone/min per mg of protein at pH 9.0 and 25°. The purification depends upon stabilization of the enzyme in glycerol-water mixtures; removal of nucleic acids with protamine sulfate; ammonium sulfate and acetone fractionations; chromatography on carboxymethyl-cellulose; and gel filtration on Sephadex G-100. The enzyme reacts also with the thionicotinamide, the 3-pyridinealdehyde, and the acetylpyridine analogs of nicotinamide-adenine dinucleotide. The effect of pH and temperature on the reaction have been measured. The application of the enzyme to the sensitive enzymatic assay of androsterone is illustrated.
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