TRANSPORT OF CITRATE CATALYZED BY THE SODIUM-DEPENDENT CITRATE CARRIER OF KLEBSIELLA-PNEUMONIAE IS OBLIGATORILY COUPLED TO THE TRANSPORT OF2 SODIUM-IONS

Aerobically grown Escherichia coli GM48 harboring plasmid pKScitS that codes for the sodium-dependent citrate carrier from Klebsiella pneumo niae (CitS) allows initial-rate measurements of citrate uptake in whol e cells. The cation stoichiometry and selectivity of CitS was studied using this experimental system. The relationship between the initial r ate of uptake of citrate and the Na+ concentration was sigmoidal at pH values between 5 and 7 suggesting a Na+ stoichiometry higher than 1. Rates of uptake increased quadratically in a range of non-saturating N a+ concentrations showing that two Na+ are translocated/catalytic cycl e. Symport of Na+ is absolutely required in the range pH 5-7 because n o uptake could be detected in the absence of Na+. Protons cannot repla ce Na+ in the translocation step but the decrease in apparent affinity for Na+ towards lower pH suggests that protons can compete with Na+ f or the cation-binding sites. Li+ can replace Na+ in the symport reacti on but it takes about a 200-fold higher concentration of Li+ over Nato achieve the same rate of uptake, showing that the affinity of CitS for Li+ is much lower than for Na+. Though high Lif concentrations hav e an inhibitory effect on citrate uptake, the data suggest that the Li + stoichiometry is also 2.