Use of the Synthetic Peptide Neurogranin(28-43) as a Selective Protein Kinase C Substrate in Assays of Tissue Homogenates

✍ Scribed by A. Gonzalez; E. Klann; J.S. Sessoms; S.J. Chen

• Publisher: Elsevier Science
• Year: 1993
• Tongue: English
• Weight: 518 KB
• Volume: 215
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1993.1574

✦ Synopsis

The synthetic peptide neurogranin ({ }{(28-43)}), the sequence of which is homologous to the phosphorylation site of the brain specific protein kinase (C) (PKC) substrates neurogranin and neuromodulin, was tested for its utility as a PKC substrate in crude tissue homogenates. The phosphorylation of neurogranin (28-43) shows time- and protein concentration-dependency. In prolonged incubations, the addition of the protein phosphatase inhibitor sodium pyrophosphate results in increased phosphorylation of neurogranin (28-43) . The phosphorylation of neurogranin (28-43) (^{\text {, was compared to }}) that of another widely used PKC substrate, (\mathbf{S 6}{(229-249)}). Neurogranin ({ }{(28-43)}) is as potent as (\mathbf{S 6}{(\mathbf{2 2 9 - 2 4 8 )}}) and more selective than (\mathbf{S 6}{(229-249)}) as a (\mathrm{PKC}) substrate. Greater than (95 %) of phosphate incorporation into neurogranin ({(28-43)}) can be inhibited by a selective PKC inhibitor, (\mathbf{P K C}{(19-38)}). Kinetic analysis of neurogranin ({ }{(28-43)}) phosphorylation in hippocampal homogenate revealed an apparent (K_{m}) of (147 \mathrm{nM}), virtually identical to previously published (\boldsymbol{K}{m}) observed for phosphorylation of the substrate by purified PKC. In addition, we assayed several neuronal and nonneuronal tissues using neuro(\operatorname{granin}{(\mathbf{2 8 - 4 3 )}}) as substrate in the presence or absence of detergent. We show that the relative PKC activity assayed with neurogranin (\mathbf{( 2 8 - 4 3 )}) ) correlates well to the relative amount of PKC known to be present in various neuronal and nonneuronal tissues. Overall, this report shows that neurogranin ({ }_{(28-43)}) can be used to selectively assay PKC, even in tissue containing low PKC activity. 1994 Academic Press, Inc.