Endogenous protein phosphorylation in chick and rat brain synaptic membranes

Abstract

The protein kinase activities endogenous to synaptic membranes prepared by an identical procedure from avian (chick) and mammalian (rat) brains were compared. Both species showed similar responses towards both protein kinase effector molecules cyclic adenosine monophosphate and Ca 2+. K app for cyclic adenosine monophosphate-dependent protein kinase activity occurred at 0.4–0.8μM cAMP and K app for Ca 2+-dependent, calmodulin-requiring protein kinase activity occurred at 1–2μM Ca 2+ (free ion concentration) both in the absence or presence of calmodulin added to the reaction mixture. This suggests that endogenous calmodulin in these membranes was able to modulate the Ca 2+-dependent, calmodulin requiring protein kinase activity. After EGTA-treatment of the membranes to remove endogenous Ca 2+ and calmodulin, no significant response towards Ca 2+ on the phosphorylation of the membrane polypeptides was measured unless exogenous calmodulin was added after which the K app for Ca 2+ was increased to 15μM Ca 2+ (free ion concentration). There was a difference in the maximal levels of kinase activity in these membranes with chick membranes containing 57% less cyclic adenosine monophosphate-dependent protein kinase activity, but 65% more Ca 2+-dependent, calmodulin-requiring protein kinase activity than the rat membranes. Similar results were determined when either low (5 μM) or high (5.8mM) concentrations of adenosine 5′-triphosphate were added to the reaction mixtures. Besides certain species differences in the molecular weights of the resulting phosphoproteins, we observed several major differences with respect to the absence or presence of some of the phosphoproteins. Chick synaptic membranes may lack the cyclic adenosine monophosphate-requiring, microtubule-associated phosphoprotein, MAP 2, one of the 2 neurospecific, cyclic adenosine monophosphate-requiring and Ca 2+, calmodulin-requiring phosphoproteins (Protein Ib, although Protein Ia apparently is present), and the Ca 2+-requiring, calmodulin-independent, ACTH-sensitive phosphoprotein, B50. The phenothiazines, trifluoperazine, fluphenazine and chlorpromazine were found to inhibit the Ca 2+-dependent, calmodulin-requiring protein kinase activities of both the chick and rat synaptic membranes. This inhibition appeared to be specific for calmodulin because at the same concentrations the phenothiazine analogue, chlorpromazine-sulfoxide, had no effect on this activity. Also found to inhibit Ca 2+-dependent calmodulin-requiring protein kinase activity were dibucaine and adrenocorticotropin. These data suggest that rat forebrain synaptic plasma membranes are activated by cyclic adenosine monophosphate to a greater extent than are chick forebrain synaptic plasma membranes while the chick membranes are activated to a greater extent by Ca 2+, calmodulin than are the rat membranes, though similarities dominate comparison of the two membrane systems.