Mechanisms of Active Transport in Isolated Membrane Vesicles

• Published in: The Journal of biological chemistry 1971-09, Vol.246 (17), p.5518-5522
• Main Authors: Barnes, Eugene M., Kaback, H.R.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)61936-2

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 > NADH = d -lactate > l -lactate) and the ability of these compounds to stimulate lactose transport ( d -lactate >> succinate > l -lactate > 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 b 1 . 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.