Abstract
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 DEGREES, RESPECTIVELY. The eft 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 Ca2+:Pi ratio of approximately 1.5 was found, suggesting formation of Ca3(PO4)2. Monovalent cations stimulated calcium uptake, with the order of effectiveness being K+ is greater than Na+ is greater than Li+ is greater than NH4+. Inhibition was found with certain divalent cations, but these also inhibited the electron transport chain. Of the divalent cations examined only Mg2+ and Sr2+ 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 Km of 0.34 mM and a Vmax of 85 nmol/min/mg of protein. The kinetic constants of the high affinity system were 4.5 muM and 2 nmol/min/mg of protein. The energy for calcium transport could be derived from the electron transport chain by oxidation of NADH, D-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 enerty for calcium transport. The ATP-driven reaction was inhibited by inhibitors of the Mg2+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.
Highlights
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
Membrane vesicles prepared by the osmotic lysis of sphero- energy coupling for active transport and other energy-requiring plasts of Escherichia coli and other bacteria have been used extensively for the study of active transport (1)
Effect of Lysis Pressure on Calcium Transport in Inverted Vesicles-A major difficulty in the preparation of inverted vesicles is the low yield of membranes obtained using the French press at 4,000 p.s.i
Summary
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. ATP can drive transport in cells (3, 4), while other data indicate that the vesicles are a whole cells (lo), it has been suggested that an indirect mixture of right-side-out and inverted membranes after freeze- mechanism exists, rather than direct coupling via the thawing (4) or even when freshly prepared (5, 6). The purpose of this report is to extend the observations described in our earlier paper (12) and to investigate the conditions for preparation and storage of inverted vesicles, and of calcium transport in these vesicles
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