Protein kinase C and dopamine release—I: Measurement by thiophosphorylation

To examine the hypothesis that protein kinase C (PKC) plays a role in the release of dopamine (DA) in the nigrostriatal pathway, a new thiophosphorylation procedure was developed to monitor PKC activity. In this method, tissues were incubated with adenosine 5'-[γ-thio 35S]triphosphate, and the transfer of the γ-thiophosphoryl group to histones or endogenous substrate proteins was measured. The thiophosphorylation showed a marked dependency on both calcium and lipids, and the endogenous substrate proteins being thiophosphorylated were similar to those reported as being specific substrates of PKC using [ 32P]ATP. Furthermore, the thiophosphorylation activity measured in the presence of calcium and lipids did not reflect cAMP-dependent or calmodulin-dependent protein kinase activities. Besides providing an accurate measure of PKC activity, thiophosphorylation has the advantage that it measures a phosphorylating activity that is independent of phosphatase activity because the thio-phosphorylated substrates are resistant to the action of phosphatases.