The effect of carbonyl cyanide m-chlorophenylhydrazone on steroid transport in membrane vesicles of Pseudomonas testosteroni

Abstract

The uncoupler carbonyl cyanide chlorophenylhydrazone (CCCP) was an effective inhibitor of steroid transport in membrane vesicles of Pseudomonas testosteroni between 10 μM and 1 mM CCCP. At these concentrations the inhibition of steroid transport was not due to an inhibition of the 3β and 17β-hydroxysteroid dehydrogenase enzyme. CCCP also affected testosterone-dependent oxygen consumption at concentrations up to 100 μM and inhibited respiration at 0.5 and 1 mM. The effect of CCCP on testosterone-dependent oxygen consumption indicated that CCCP was acting as an uncoupler. The concurrent inhibition of testosterone transport and stimulation of testosterone-dependent oxygen consumption at 10–100 μM CCCP supported the conclusion that transport and metabolism were tightly coupled processes. When membrane vesicles were pre-incubated with CCCP for 15 min, CCCP did inhibit transport and the 3β and 17β -hydroxy steroid dehydrogenase activity. However, both transport and enzyme inhibition could be prevented by the addition of NAD + to the incubation mixture. This indicated that CCCP exhibits the properties of a sulfhydryl reagent under pre-incubated conditions.