Rat liver 3-oxo-5 alpha-steroid delta 4-dehydrogenase. Modulation of enzyme activity by changes in phosphorylation...

3-Oxo-5 alpha-steroid: NAD+ delta 4-oxidoreductase ("NADH-5 alpha-reductase", EC 1.3.1.?) is rapidly inactivated in the presence of 17 beta-hydroxy-4-androsten-3-one (testosterone). This activation is prevented by increasing the phosphate concentration. When the enzyme assay is carried out in Tris-HCl, only a small activity (1.7 nmol X min-1 X mg-1) is observed which may be further decreased by addition of phosphatases. Addition of the phosphatase inhibitor dextran sulphate or ATP, Mg++ and c-AMP results in a significant increase of activity (228% and 273%, respectively) compared with the Tris-HCl control. Glycerol 2-phosphate and glycerol 3-phosphate have a stabilizing effect on 3-oxo-5 alpha-steroid: NAD+ delta 4-oxidoreductase by decreasing the Km towards the substrate testosterone from 1.2 X 10(-5) mol/l to 3.3 X 10(-6) mol/l. V remains unchanged. Half maximal velocity (testosterone reduction) is achieved with 20 mumol/l glycerol 2-phosphate and glycerol 3-phosphate. Addition of c-AMP dependent protein kinase (EC 2.7.1.37) to a microsomal preparation pretreated with alkaline phosphatase (EC 3.1.3.1) results in a significant increase of 3-oxo-5 alpha-steroid: NAD+ delta 4-oxidoreductase activity compared with the control.