Purification and characterization of an ATP-binding protein from brain membranes

An ATP-binding protein was purified from pig cerebral cortex. Extraction of a crude membrane fraction with distilled water, differential acetone fractionation and sequential column chromatography on Sephadex G 200, DEAE-Sephacel and an ATP-affinity Sepharose led to a 310-fold purification. ATPase, phosphodiesterase, adenylate cyclase and protein kinase were separated during the preparation. ATP-binding was studied by a filtration technique. Specific binding of ATP exhibited a biphasic time course and was pH and temperature dependent. Scatchard plots of equilibrium binding data were nonlinear suggesting the existence of two different binding sites for ATP. The dissociation constants (K(d)) were 4.1 x 10(-8) and 2.0 x 10(-7) M and the maximal number of binding sites (B(max)) 0.68 and 1.0 nmol ATP bound per mg of purified protein. Several related compounds were studied for their ability to inhibit ATP-binding. Only ADP, GTP and CTP, but not adenosine, AMP, cyclic AMP and cyclic GMP, were potent in displacing ATP from its binding sites. Specific binding of ATP was strictly dependent on the presence of divalent cations, Mg(2+), Mn(2+) and Ca(2+) being equipotent.