TRANSPORT OF CITRATE CATALYZED BY THE SODIUM-DEPENDENT CITRATE CARRIER OF KLEBSIELLA-PNEUMONIAE IS OBLIGATORILY COUPLED TO THE TRANSPORT OF 2 SODIUM-IONS

Aerobically grown Escherichia coli GM48 harboring plasmid pKScitS that codes for the sodium-dependent citrate carrier from Klebsiella pneumoniae (CitS) allows initial-rate measurements of citrate uptake in whole cells. The cation stoichiometry and selectivity of CitS was studied using this experimental system. The relationship between the initial rate 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 Na+ concentrations showing that two Na+ are translocated/catalytic cycle. Symport of Na+ is absolutely required in the range pH 5-7 because no uptake could be detected in the absence of Na+. Protons cannot replace Na+ in the translocation step but the decrease in apparent affinity for Na+ towards lower pH suggests that protons can compete with Na+ for the cation-binding sites. Li+ can replace Na+ in the symport reaction but it takes about a 200-fold higher concentration of Li+ over Na+ to achieve the same rate of uptake, showing that the affinity of CitS for Li+ is much lower than for Na+. Though high Lif concentrations have an inhibitory effect on citrate uptake, the data suggest that the Li+ stoichiometry is also 2.