Use of the synthetic peptide neurogranin((28-43)) as a selective protein kinase C substrate in assays of tissue homogenates

A. Gonzalez, Eric Klann, J. S. Sessoms, S. J. Chen

Research output: Contribution to journalArticle

Abstract

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 K(m) of 147 nM, virtually identical to previously published K(m) 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.

Original languageEnglish (US)
Pages (from-to)184-189
Number of pages6
JournalAnalytical Biochemistry
Volume215
Issue number2
DOIs
StatePublished - 1993

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Protein Kinase C
Assays
Phosphorylation
Tissue
Peptides
Substrates
S 6
EphA5 Receptor
Neurogranin
neurogranin (28-43)
GAP-43 Protein
Protein C Inhibitor
Phosphoprotein Phosphatases
Protein Kinase Inhibitors
Detergents
Brain
Phosphates
Kinetics

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Use of the synthetic peptide neurogranin((28-43)) as a selective protein kinase C substrate in assays of tissue homogenates. / Gonzalez, A.; Klann, Eric; Sessoms, J. S.; Chen, S. J.

In: Analytical Biochemistry, Vol. 215, No. 2, 1993, p. 184-189.

Research output: Contribution to journalArticle

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abstract = "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 K(m) of 147 nM, virtually identical to previously published K(m) 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.",
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