ON THE 3α‐HYDROXYSTEROID DEHYDROGENASE FROM PSEUDOMONAS TESTOSTERONI. The effect of denaturing agents

Abstract

Highly purified preparations of the 3α-hydroxysteroid:NAD- oxidoreductase (E.C.1.1.1.50) from Pseudomonas testosteroni (ATCC 11996) which consist of two major isoenzymes, with traces of a third, have been split into two enzymatically inactive polypeptides A and B by the use of sodium dodecylsulphate, urea and guanidinium hydrochloride. Both polypeptides have a molecular weight of 25,000 ± 2,500 as shown by thin-layer gel chromatography and ultracentrifugations. They differ, however, in charge as shown by electrophoresis on cellulose acetate strips in the presence of 8 M urea. Each of the isoenzymes, have molecular weight of about 50,000 and thus consist of two subunits. The presence of the three isoenzymes may be explained by the following combinations of the subunits AA, AB and BB. Close to 100% of the original activity towards the three substrates, androsterone, tetrahydrocortisone and desoxycholate could be restored within 24 h when the inactivated enzyme was diluted in order to remove the effect of the denaturant.