Phosphate binding by cerebral microsomes in relation to adenosine-triphosphatase activity

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Abstract
Microsomes prepared from guinea-pig and ox brain were incubated for periods of a few seconds with low concentrations of Mg-[32P]ATP, the reaction was stopped with trichloroacetic acid and determinations were made of the phosphate bound to the acid-washed, and in some cases solvent-extracted, residue. At 20 [mu][image]-ATP, at 37[degree] and in the presence of Na+ ions, 30-50 [mu]moles of phosphate/mg. of microsomal protein were bound by the preparation within 1 sec. of starting the reaction; little further change in level occurred until hydrolysis of ATP exceeded 50%, when the bound phosphate began to decline fairly rapidly to the zero-time value. At 20 [mu][image]-ATP without Na+ ions present or in the presence of K+ ions, the level of bound phosphate increased gradually and did not decline as ATP hydrolysis approached completion. Potassium ions either inhibited the formation of Na+-dependent bound phosphate or, when added during the course of the reaction, rapidly reduced its level. At 200 [mu]M[image]-ATP the bound phosphate formed in the presence of Na+ ions appeared to consist of a mixture of the unstable Na+-dependent type and the stable type requiring only Mg2+ ions for its formation. Nonradioactive ATP added during the course of the reaction at 20 [mu][image]-ATP with Na+ ions present rapidly discharged virtually all the bound 32P counts; at 200 [mu][image]-ATP only a proportion of the label was similarly discharged. The Na+ -dependent bound phosphate is therefore turning over in contrast with that formed in the absence of Na+ ions, which proved more stable. The Na+ -dependent bound phosphate was not in the form of ATP; experiments with [14C]ATP instead of [32P]ATP showed a small and invariable binding of ATP by the preparation unaffected by Na+ ions or time of incubation. Under the usual conditions employed in this work ouabain stimulated formation of Na+-dependent bound phosphate when Na+ ions were suboptimum and inhibited it when optimum Na+ ions were present. The Na+-dependent binding reaction under present conditions did not invovle incorporation into phosphorylserine groups. The relation of the fingings to the (Na+,K+)-ATPase of the preparation and to observations in brain slices appearing to implicate phosphorylserine groups in cation transport, is discussed.