Endogenous phosphorylation of rat brain synaptosomal plasma membranes in vitro: some methodological aspects

The time course of endogenous phosphorylation in vitro of total or separated synaptic plasma membrane proteins (SPM) has been correlated with that of hydrolysis of the phosphate donor (ATP) in the incubation medium. The ATP/SPM ratio in the medium was varied. In a low-ratio medium (7.5 muM ATP; 2.2 microgram SPM/microliter) a complete hydrolysis of ATP occurred almost instantaneously as was measured by the release of free phosphate in and the disappearance of ATP from the medium. As a consequence, only a very short peak of phosphorylation, followed by dephosphorylation was observed. However, when higher ATP/SPM ratios were used (200 muM ATP; 0.4 microgram SPM/microliter and 500 muM ATP; 0.4 microgram SPM/microliter), the incorporation of phosphate into SPM proteins was linear for 20 sec, and the maximum level of phosphate incorporation was increased. Similar results were obtained after separation of 32P-labeled phosphoproteins by slab gel electrophoresis. However, analysis of the autoradiographs obtained from one SPM preparation under different ATP/SPM ratios revealed dependence of phosphorylation of individual protein bands on the conditions used.