Mechanism of glutamate transport in Escherichia coli B. 1. Proton-dependent and sodium ion dependent binding of glutamate to a glutamate carrier in the cytoplasmic membrane.
Specific binding of glutamate to its carrier was investigated by using cytoplasmic membrane vesicles prepared from Escherichia coli B. The binding activity was specifically affected by the Na+ and H+ concentrations of the medium. Cytoplasmic membrane vesicles from the mutant strain 36-39 that is defective in the Na+-dependent glutamate transport system showed no binding of glutamate. Addition of the protonophore uncoupler 3,5-di-tert-butyl-4-hydroxy-benzylidenemalononitrile or carbonyl cyanide m-chlorophenylhydrazone, or the ionophore monensin or nigericin, did not inhibit the binding, indicating that the binding reaction is not energy dependent. The parameters of binding were determined in reaction media with various combinations of H+ and Na+ concentrations. The maximum number of binding sites was constant and determined to be 70 pmol/mg of membrane protein, irrespective of the concentrations of H+ and Na+ in the medium. The apparent dissociation constant, however, was greatly affected by changes in the concentrations of both H+ and Na+, in such a way that it was expressed by a linear combination of the reciprocals of the H+ and Na+ concentrations. The characteristics of binding can be explained best by supposing that glutamate can bind only to a H+/Na+/carrier complex that is formed by random binding of H+ and Na+ to the unloaded carrier. The physiological role of this elementary binding reaction and of this quaternary complex as an active intermediate in the process of glutamate transport is discussed.
D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D007700:Kinetics; D008433:Mathematics; D012965:Sodium Chloride / Q000494:pharmacology*
D002352:Carrier Proteins; D005971:Glutamates; D018698:Glutamic Acid; D012965:Sodium Chloride