The sodium-stimulated adenosine-triphosphatase activity and other properties of cerebral microsomal fractions and subfractions

Abstract

By differential centrifuging of sucrose dispersions of guinea-pig cerebral cortex, 75% of their Na-stimulated adenosine-triphosphatase activity was obtained in a microsomal fraction; the tissue activity was computed as 1100-1500 umoles of phosphate formed/g. of fresh tissue/ hr. Assay conditions for the enzyme, and changes in its activity on keeping, are described. The optimum pH was 7.0-7.4; half-maximal activity with 3 mM-adenosine triphosphate, 3 mM-Mg and 30 mM-K was given by 15 mM-Na. With 3 mM-Mg, 30 mM-K and variable adenosine triphosphate, extrapolation suggested half-maximal activity at 20 mM-ATP; with 100 mM-Na in addition, the value became 8.7 mM-ATP. Differential and density-gradient centrifuging indicated the enzyme system to be associated with the membrane structures rather than the ribonucleic acid granules of the microsomal fraction. Fractions richest in the adenosine triphosphatase possessed negligible succinic dehydrogenase but were enriched in acetylcholinesterase and in cholesterol. A proportion of the adenosine-triphosphatase activity was no longer deposited at 6.2 x 106 g-min. after treatment of microsomal fractions with deoxycholate, with Lubrol W or with digitonin; with the last-named reagent stimulation by Na was retained to an appreciable degree by the enzyme of the supernatant. The adenosine-triphosphatase activity of untreated microsomal fractions was inhibited by 2,4,6-trinitrobenzenesulphonate and by suramin, in reaction mixtures with Mg and K, or with Mg, K and Na salts. Ouabain at 10-7-10-4 M inhibited with Mg, K and Na but not without Na. Basic polypeptides activated with Mg and K but not with Mg, K and Na. Competition was noted between protamine, Mg and alkali metals for salt formation with adenosine triphosphate. The data are discussed in relation to the likely functioning of the enzyme system in active Na and K transport.