Purification and Properties of 17β-Hydroxy-C19-Steroid Dehydrogenases of Adult Male Guinea Pig Kidney*

Abstract

The 17.beta.-hydroxy-C19-steroid dehydrogenase activity of adult male guinea pig kidney was separated into 1 isolated and 2 contiguous enzymatically pure fractions by submitting the cytosol to (NH4)2SO4 (40-80% saturation) precipitation, Sephadex G-75 filtration and DEAE-Sephadex A-50 and CM-Sephadex C-50 chromatography. Further DEAE-Sephadex A-50 and CM-Sephadex C-50 chromatography separated 8 isozymes, which were divided into 4 groups in accordance with their behavior on chromatography and mobility on gel electrophoresis. The MW of the purified enzymes were identical by Sephadex filtration (33,000) and SDS gel electrophoresis (34,000). Two of the enzymes were separated by SDS gel electrophoresis into 2 subcomponents of 23,000 and 11,000 daltons. The amino acid composition, Km values and coenzyme and substrate specificities of the enzymes (except 1) were very similar. The pI [isoelectric point] values varied from 5.1-6.4. HgCl2 and PCMB [p-chloromercuribenzoate] inhibited enzyme activity, but prior addition of cysteine prevented the inhibition. The presence of phosphate or pyrophosphate greatly enhanced the trace of DPN+-linked activity. The heterogeneity was due to at least 2 factors. Rapid (within 3 h) preparation of the cytosol in 7 mM 2-mercaptoethanol yielded 1 intense and 1 minor enzyme band on gel electrophoresis. Omission of the mercaptoethanol resulted in the appearance of 3 enzyme bands, which were reversible on the addition of 2-mercaptoethanol. Storage of the cytosol at 4 or -20.degree. C in the absence or presence of 7 mM 2-mercaptoethanol and of the kidney before extraction also resulted in the exhibition of enzyme bands which were not reversible on addition of 2-mercaptoethanol. The purified enzymes exhibited only a single form on storage at -20.degree. C with 2-mercaptoethanol, but multiple forms appeared when the mercaptoethanol was removed by dialysis. The multiple forms were reverted to a single form on addition of mercaptoethanol.