Cupric ion-mediated active transport of amino acids in membrane vesicles of Mycobacterium phlei.

Abstract

In the absence of substrate oxidation, membrane vesicles of Mycobacterium phlei were shown to accumulate proline, glutamine, and glutamic acid mediated by Cu”+. The Cu’+- mediated uptake of amino acids was found to be an active process and required the presence of amino acid binding protein(s). Thus, membrane vesicles which lack active transport of a particular amino acid because of the loss of specific binding protein(s) do not accumulate these amino acids even in the presence of Cu+. The Cu’+-mediated uptake of proline exhibited a specific requirement for Na+. The Cu”--mediated uptake process was not inhibited by anaerobiosis and respiratory inhibitors, such as sodium azide and m-chlorocarbonylcyanide phenylhydrazone. How- ever, atebrin and o-phenanthroline were found to inhibit the uptake process. The results with various sulfhydryl blocking agents suggests that oxidation of available sulfhy- dry1 residues of the membrane protein(s) is required for the Cu”+-mediated uptake process. The uptake of amino acid was inhibited by p-chloromercuribenzene sulfonic acid or X-ethylmaleimide when added prior to the addition of Cu’+. In addition, efflux of amino acids, previously accumulated to the steady state level occurred when sulfhydryl reducing agents such as glutathione, cysteine, or dithiothreitol were added. Evidence is presented that disulfide configuration of the carrier or membrane protein(s) favors the transport of amino acids against the concentration gradient, whereas the sulfhydryl state of these proteins induces the efflux process. Studies show that there was 1:l stoichiometry for the oxidation of available sulfhydryl residues in the mem- brane to the reduction of Cu”, and the proton gradient generated as a result of the oxidation of sulfhydryl groups is presumably the driving force for the uptake of these amino acids. Numerous studies have been carried out to delineate the * This work was supported by grants from the National Science Foundation (BM572-018961, the National Institutes of Health, United States Public Health Service (AI 056371, the Hastings Foun- dation at the University of Southern California, School of Medicine and Bioscience Laboratories fellowship to A. J. J. The costs of publication of this article were defrayed in