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

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Abstract
The time course of endogenous phosphorylation in vitro of total or separted 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 μM ATP; 2.2 μg SPM/μl) 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 μM ATP; 0.4 μg SPM/μl and 500 μM ATP; 0.4 μg SPM/μl), 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 of32P-labeled phosphoproteins by slab gel electrophoresis. However, analysis of the autoradiographs obtained fromone SPM preparation under different ATP/SPM ratios revealed dependence of phosphorylation of individual protein bands on the conditions used.