Use of the synthetic peptide neurogranin(28-43) as a selective protein kinase C substrate in assays of tissue homogenates.
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) was compared to that of another widely used PKC substrate, S6(229-249). Neurogranin(28-43) is as potent as S6(229-249) and more selective than S6(229-249) as a PKC substrate. Greater than 95% of phosphate incorporation into neurogranin(28-43) can be inhibited by a selective PKC inhibitor, PKC(19-36). Kinetic analysis of neurogranin(28-43) phosphorylation in hippocampal homogenate revealed an apparent Km of 147 nM, virtually identical to previously published Km observed for phosphorylation of the substrate by purified PKC. In addition, we assayed several neuronal and nonneuronal tissues using neurogranin(28-43) as substrate in the presence or absence of detergent. We show that the relative PKC activity assayed with neurogranin(28-43) 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.
Gonzalez
A
A
Division of Neuroscience, Baylor College of Medicine, Houston, Texas 77030.
Klann
E
E
Sessoms
J S
JS
Chen
S J
SJ
D000595:Amino Acid Sequence; D000818:Animals; D002148:Calmodulin-Binding Proteins; D008297:Male; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D051579:Neurogranin; D010446:Peptide Fragments / Q000378:metabolism; D010764:Phosphorylase Kinase / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity
D002148:Calmodulin-Binding Proteins; D009419:Nerve Tissue Proteins; C496651:Nrgn protein, rat; D010446:Peptide Fragments; D051579:Neurogranin; D010764:Phosphorylase Kinase; D011493:Protein Kinase C