Mechanisms of Active Transport in Isolated Membrane Vesicles: I. THE SITE OF ENERGY COUPLING BETWEEN d-LACTIC DEHYDROGENASE AND β-GALACTOSIDE TRANSPORT IN ESCHERICHIA COLI MEMBRANE VESICLES

Abstract

Abstract Transport of a wide variety of amino acids and sugars by membrane vesicles isolated from Escherichia coli ML 308-225 is coupled primarily to d-lactic dehydrogenase. This membrane-bound, flavin-linked primary dehydrogenase is coupled to oxygen via a cytochrome system also present in the vesicle membrane. Spectrophotometric evidence shows that d-lactic dehydrogenase, succinic dehydrogenase, l-lactic dehydrogenase, and NADH dehydrogenase all utilize the same cytochrome system. There is no relationship between rates of oxidation of electron donors by the respiratory chain (succinate g NADH = d-lactate g l-lactate) and the ability of these compounds to stimulate lactose transport (d-lactate >> succinate g l-lactate g NADH). Furthermore, d-lactate in combination with other electron donors is no more effective than d-lactate alone for support of lactose transport. These findings indicate that the site of energy coupling of d-lactic dehydrogenase to active transport lies between the primary dehydrogenase and cytochrome b1. Supportive evidence for this conclusion is obtained from experiments showing that N-ethylmaleimide and p-chloromercuribenzoate inhibit transport and d-lactate-induced respiration. However, these sulfhydryl reagents do not inhibit d-lactic dehydrogenase with dichlorophenolindophenol as an artificial acceptor, nor do they significantly block NADH-induced respiration.