THE EFFECT OF NEUROCATIN ON PROTEIN-PHOSPHORYLATION IN STRIATAL SYNAPTOSOMES FROM RAT-BRAIN

Neurocatin, a neuroregulatory factor isolated from mammalian brain, is a powerful affector of protein phosphorylation in rat striatal synaptosomes. Two major synaptosomal phosphoproteins of approximately 80 and approximately 60 kDa, possibly synapsin I and tyrosine hydroxylase, were especially sensitive to neurocatin. Immunoprecipitation experiments confirmed that the 60-kDa protein is the enzyme tyrosine hydroxylase. At low concentrations of neurocatin (to approximately 7.5 ng/100 mul of suspension), incorporation of P-32 orthophosphate into these proteins increased with increasing neurocatin concentration. At 7.5 ng of neurocatin, incorporation of the label into the two proteins increased by 22 and 26%, respectively. Concentrations of neurocatin > 7.5 ng/100 mul caused progressive decrease in incorporation of P-32 into many synaptosomal proteins; by a concentration of neurocatin of approximately 45 ng/100 mul, the level of P-32 incorporation into many proteins was less-than-or-equal-to 70% of control. The effects of neurocatin on synaptosomal protein phosphorylation were also dependent on the time of incubation. At a constant concentration of approximately 7.5 ng/100 mul of neurocatin, increased incorporation of P-32 into many proteins was measurable within 0.5 min and was maximal by 1 min. Incubation times > 2.0 min, showed progressive decrease in P-32 incorporation. Removing extrasynaptosomal Ca2+ with EGTA attenuated the increased P-32 incorporation induced by low neurocatin concentrations, suggesting that calcium plays a role in neurocatin-induced phosphorylation of rat striatal synaptosomal proteins. The reduced incorporation of label induced by high neurocatin concentrations, however, was not calcium dependent. The effects of neurocatin on the level of P-32 incorporation into proteins were observed only in intact synaptosomes, consistent with this compound acting through receptors on the plasma membrane.