MECHANISM OF PROLINE TRANSPORT IN ESCHERICHIA-COLI-K12 .3. INHIBITION OF MEMBRANE POTENTIAL-DRIVEN PROLINE TRANSPORT BY SYN-COUPLED IONS AND EVIDENCE FOR SYMMETRICAL TRANSITION-STATES OF THE 2H+/PROLINE SYMPORT CARRIER

Abstract

Specific inhibition of 2H+/proline symport by syn-coupled ions (Na+, Li+ and H+) was investigated using cytoplasmic membrane vesicles prepared from the proline carrier-overproducing strain MinS/pLC4-45 of E. coli K12. The 2H+/proline symport driven by the membrane potential generated via respiration with 20 mM ascorbate/Tris, 0.1 mM phenazine methosulfate was specifically inhibited by Na+. The inhibition by Na+ was described by a fully noncompetitive mechanism, and the apparent Ki for Na+ was 15 mM. A linear correlation between the apparent Vmax and the apparent Kd was observed. Li+ stimulated the transport activity 2-fold at 10 mM and inhibited it at concentrations > 50 mM. H+ caused fully noncompetitive inhibition of 2H+/proline symport, and its apparent Ki was 0.6 .mu.M. Evidently, the concentrations of Na+ and H+ strictly and independently regulate the amount of the active C state carrier responsible for 2H+/proline symport driven by the membrane potential by inhibiting the transition from the C* state carrier which exhibits Na+- and H+-dependent binding of proline and is predominant in nonenergized conditions.