ATP Splitting and Calcium Binding by Brain Microsomes Measured with a Rapid Perfusion Method

Abstract

Rat brain microsomes, immobilized on a filter, were perfused with ATP-containing solutions in a device which made possible rapid change of perfusion media and frequent sampling of effluent. Inorganic phosphate production could be measured 10 times per sec. When ATP, sodium, or potassium was absent from the first perfusion medium and present in a second, and introduced without interrupting flow, phosphate output rose within a few tenths of a second. Inhibition by ouabain began within 0.3 sec but did not become maximal for at least 10 sec. Rapid binding of ouabain was minimal or absent, as was rapid release of ouabain on introducing potassium abruptly. Although the preparation bound some calcium reversibly, no measurable uptake of calcium occurred coincident with activation by ATP or by potassium, and no measurable release of calcium occurred coincident with the onset of ouabain inhibition. However, activation by sodium was consistently associated with simultaneous release (within < 1 sec) of calcium, averaging 46 pmole per mg of protein. Calcium release in response to sodium also occurred in the absence of ATP or in the presence of ouabain. At 0°C sodium produced neither activation nor calcium release. The results are consistent with the possibility that sodium and calcium are competitively bound, even in the absence of ATP, to an active site on the enzyme distinct from the sites of potassium activation or glycoside inhibition.