Reactive exofacial sulfhydryl-groups on the arginine-ornithine antiporter of Lactococcus lactis

The effect of various sulfhydryl (SH)-specific reagents on arginine-ornithine antiport activity in membrane vesicles of Lactococcus lactis was studied. Little or no inhibition of arginine-ornithine exchange was observed with maleimides and arsenicals. HgC! 2 and the organic mercurials p-chloromercuribenzene sulfonate (pCMBS), p-chloromercuribenzoate (pCMB), and O-(3-hydroxymercuri-2-methoxypropyl)carbamylphenoxyacetate completely inactivated the antiporter. This effect could not be attributed to vesicle disruption. Inactivation of arginine-oruithine exchange by pCMBS could be reversed by dithiothreitol. It was reflected by a decreased Vm~ x with no change in the K t for arginine uptake, and correlated with a reduction of the number of arginine binding sites. The poorly penetrating p CMBS had at low concentrations only access to the reactive SH-group(s) from the outer surface of the membrane, while the permeant p CMB reacts with SH-group(s) at both membrane surfaces. Arginine and ornithine, if present on the outer surface of the membrane, protected the arginine-ornithine antiporter against p CMBS inactivation. Membrane-impermeable oxidizing agents have no effect on the exchange activity, indicating that the antiporter is not regulated by the redox potential of the environment. It is concluded that the arginine-ornithine antiporter contains one or more reactive exofacial SH-groups which are presumably located in or near the substrate binding site.