Solubilization of a phosphoprotein and its associated cyclic AMP-dependent protein kinase and phosphoprotein phosphatase from synaptic membrane fractions, and some kinetic evidence for their existence as a complex

The kinetic data obtained for the endogenous phosphorylation and endogenous dephosphorylation of Protein II, in both the Triton extract and the NH4Cl extract, are compatible with the idea thatprotein II, its cyclic AMP-dependent protein kinase, and its cyclo-AMP- dependent protein phosphatase, may exist in, and be extractable from, synaptic membranes in the form of a complex.

Abstract

Synaptic membrane preparations contain a protein with an apparent molecular weight of 49,000, designated Protein II, whose endogenous phosphorylation and dephosphorylation are regulated by cyclic AMP. Protein II and the enzymes which catalyze the cyclic AMP-dependent phosphorylation and dephosphorylation of Protein II have been obtained in a soluble form from these synaptic membrane preparations by treatment with either 0.25% Triton X-100 or 0.1 m NH4Cl. In the Triton X-100 solubilized enzyme system, the regulation of the endogenous phosphorylation of Protein II by cyclic AMP was similar to that observed in untreated synaptic membrane preparations. Thus, the effect of cyclic AMP on the phosphorylation of Protein II was stimulatory in the presence of Mg2+ and inhibitory in the presence of Zn2+. The stimulation by cyclic AMP of the phosphorylation of Protein II observed in the presence of Mg2+ could be converted to an inhibitory effect either by increased ionic strength, by Zn2+, or by a thiol reagent, dithionitrobenzoate. In the NH4Cl-solubilized enzyme system, cyclic AMP-dependent phosphorylation was also observed, but the mode of regulation by cyclic AMP of the phosphorylation of Protein II was partially altered: the effect of cyclic AMP was inhibitory in the presence of either Mg2+ or Zn2+. In both the Triton X-100-solubilized and the NH4Cl-solubilized enzyme systems, cyclic AMP facilitated the endogenous dephosphorylation of Protein II in the absence of any divalent cation, as it did in untreated membrane preparations. The kinetic data obtained for the endogenous phosphorylation and endogenous dephosphorylation of Protein II, in both the Triton extract and the NH4Cl extract, are compatible with the idea that Protein II, its cyclic AMP-dependent protein kinase, and its cyclic AMP-dependent protein phosphatase, may exist in, and be extractable from, synaptic membranes in the form of a complex.