Phosphorylation of Synaptic Membranes

: In vivo and in vitro phosphorylation of synaptic membrane proteins were compared. In vivo phosphorylation was carried out by injecting rats intraventricularly with 1 mCi of 32Pi‐orthophosphate. After a 40‐min isotope incorporation period, rats were decapitated and synaptic membranes isolated. In vitro phosphorylation was accomplished by incubating unlabeled synaptic membranes, isolated from noninjected rats, with 5 μM‐[γ‐32P]ATP. In vivo and in vitro32P‐labeled synaptic membranes were then fractionated electrophoretically on an SDS‐polyacrylamide (7.5%) slab gel. The Coomassie blue protein patterns for in vivo and in vitro32P‐labeled synaptic membranes were identical. In contrast, autoradiographs of these gels showed striking differences in the pattern of phosphate incorporation. Cyclic‐AMP (10 μM) maximally stimulated) the in vitro phosphate labeling of two protein bands (80,000 and 55,000 apparent molecular weight) which were not substantially labeled by the in vivo procedure.