Characterization of an Active Transport System for Calcium in Inverted Membrane Vesicles of Escher&k coZi*

The energy-dependent uptake of calcium by inverted membrane vesicles of Escherichia coli was investigated. Methods for preparation and storage of the vesicles were devised to allow for the maximal activity and stability of the calcium transport system. The pH and temperature optima for the reaction were observed to occur at pH 8.0 and 30”, respectively. The effects of various ions on the transport reaction were determined. It was found that the extent of the reaction depended on the presence of phosphate or oxalate. Phosphate was found to enter the vesicles at a rate slower than that of calcium. A Ca*+:P, ratio of approximately 1.5 was found, suggesting formation of Ca,(PO,),. Monovalent cations stimulated calcium uptake, with the order of effectiveness being K+ > Na+ > Li+ > NH,+. Inhibition was found with certain divalent cations, but these cations also inhibited the electron transport chain. Of the divalent cations examined only Mg2+ and Sr*+ inhibited calcium transport without a corresponding inhibition of respiration. Calcium transport exhibited biphasic kinetics, with a low affinity system and a high affinity system. The low affinity system showed a K, of 0.34 mM and a V,,,,, of 85 nmol/min/mg of protein. The kinetic constants of the high affinity system were 4.5 FM and 2 nmol/min/mg of protein. The energy for calcium transport could be derived from the electron transport chain by oxidation of NADH, n-lactate, and succinate, in order of their effectiveness. Respiration-driven calcium transport was inhibited by inhibitors of the electron transport chain and by uncouplers of oxidative phosphorylation. ATP could also be used to supply energy for calcium transport. The ATP-driven reaction was inhibited by inhibitors of the Mg*+ATPase and by an antiserum prepared against that protein, demonstrating that that enzyme is involved in the utilization of ATP for active transport in inverted vesicles.