Ultracytochemical demonstration and probable localization of 3beta-hydroxysteroid dehydrogenase activity with a ferricyanide technique.

Abstract

In order to localize 3beta-hydroxysteriod dehydrogenase activity on the ultrastructural level, sections of Newt and Rat adrenocortical tissues, fixed in a mixture of glutaraldehyde (0.25%) and formaldehyde (1%), were incubated in a medium containing namely a 3beta-hydroxysteroid as substrate, NAD, potassium ferricyanide as final electron acceptor and copper sulfate. In some experiments, phenazine methosulfate (PMS), an electron carrier which can substitute for the activity of the endogenous NADH-diaphorase, is added at various concentrations to the incubation medium. A final precipitate of copper ferrocyanide is observed in the immediate vicinity of the tubules of the smooth endoplasmic reticulum, or in contact with their external faces. The reaction product can also be seen in mitochondrial cristae. The reaction does not take place in incubation media lacking substrate or containing cyanoketone, a specific inhibitor of 3beta-hydroxysteroid dehydrogenase. the addition of PMS to the incubation medium increases the intensity of the reaction, but does not modify the localization of the precipitate.