Purification and properties of a citrate-binding transport component the C protein of Salmonella typhimurium

Abstract

Salmonella typhimurium was shown to contain a citrate-binding protein (C protein) which was purified to homogeneity from the periplasmic fraction released by cold osmotic shock. The protein is dimeric, has an apparent molecular weight of 28 000 and an isoelectric point of 6.1. Sodium ions were required for optimum substrate binding, however, the divalent cations Zn2+, Mg2+, and Co2+ were inhibitory. The C protein was relatively stable but sensitive to various detergents and chaotropic agents. Approximately one citrate molecule was bound per molecule of protein and citrate binding (Kd = 1–2.6 μM) was strongly competitively inhibited by DL-isocitrate and DL-fluorocitrate but not by other carboxylates. Neither succinate, glutamate, nor acetate were bound to the C protein. No apparent enzyme activity was associated with this protein. A concomitant reduction in the level of binding protein and in citrate transport activity occurred in osmotically shocked cells as well as with L-malate- or succinate-grown cells. Fluorocitrate-resistant mutants were simultaneously defective in citrate transport, citrate binding, and production of cross-reacting material. One transport-defective mutant did produce citrate binding protein.