Active transport in Excherichia coli B membrane vesicles. Irreversible uncoupling by chloropyruvate

Abstract

In the accompanying report (Kaczorowski, G., Shaw, L., Laura, R., and Walsh, C. (1975) J. Biol. Chem. 250, 8921-8930), we have shown that the oxidation of beta-chloro-D-alanine by a membrane-bound D-alanine dehydrogenase results in the inactivation of both dehydrogenase-coupled and P-enolpyruvate-dependent active transport in membrane vesicles. We have also demonstrated that chemically prepared chloropyruvate has the same inactivating effects on transport. In this report, we show that in addition to abolishing hexose and proline uptake, chloropyruvate inhibits lactose and several other amino acid uptake systems to different extents, although proline transport is the most severely inhibited. The degree of transport inactivation also depends on whether the keto acid is added exogenously or is generated by the D-alanine dehydrogenase. Chloropyruvate treatment does not inhibit D-alanine dehydrogenase, D-lactate dehydrogenase of the passage of electrons to oxygen by the membrane cytochrome chain. However, alanine racemase and pyruvate oxidase (to a lesser extent) are inactivated by this keto acid. Treatment of vesicles with chloropyruvate does not affect the establishment of maintenance of a membrane potential, however, this does inhibit solute transport in response to an artificially induced potential. If chloropyruvate is added at any point during a time course of proline transport, there is an instantaneous blockade of amino acid uptake suggesting that the proline carrier can no longer translocate solute across the membrane. Upon examining the functionality of the carrier protein after exposure to chloropyruvate, there is no appreciable difference in efflux or exchange properties as compared to untreated controls. Therefore chloropyruvate does not block proline passage through the membrane, but rather appears to interfere with the ability of the proline carrier to sense the membrane potential. The beta-halo keto acid does not then uncouple respiration from energization of the membrane but does interfere with the ability of the energized membrane state to be used for the transport of most solutes.