The Effect of Sulfhydryl Inhibitors on Substrate Oxidation and Proline Transport with Membrane Preparations from Mycobacterium phlei

Abstract

Abstract Substrate oxidation and amino acid transport were examined in the presence of sulfhydryl reagents with membrane preparations from Mycobacterium phlei. Oxidation of exogenously added β-NADH, succinate, ascorbate N, N, N', N'-tetramethyl-p-phenylenediamine, or ascorbate phenazine methosulfate was inhibited by organic mercurials. The organic mercurials inhibited succinate oxidation between cytochrome b and c. N-ethylmaleimide did not inhibit the oxidation of succinate, ascorbate N, N,N', N'-tetramethyl-p-phenylenediamine or ascorbate phenazine methosulfate, but did block NADH oxidation at the level of NADH dehydrogenase. The rate and steady state level of proline transport was lowered slightly by N-ethylmaleimide with all substrates. The organic mercurials inhibited the uptake of proline or induced proline efflux if added after transport steady state levels had been obtained. By altering the concentration of the mercurials, it was possible to obtain concentrations which did not inhibit substrate oxidation, but inhibited proline transport. The inhibition of transport was not as great if proline was preincubated with the membrane vesicles before the addition of inhibitor. Preincubation of the membrane preparations with the organic mercurials inhibited the nonenergized accumulation of proline; preincubation with proline afforded some protection from this inhibition. Thus, the organic mercurials appear to act at the level of a protein involved in transport. The ability to dissociate proline transport from substrate oxidation suggests that with membrane preparations from M. phlei there are a minimum of two distinct sulfhydryl moieties: one group is involved with proline transport and at least one group is involved with substrate oxidation.