Peptide substrates suitable for assaying glycogen synthase kinase-3 in crude cell extracts

Anal Biochem. 1997 Jan 1;244(1):16-21. doi: 10.1006/abio.1996.9838.

Abstract

In this study we describe the characterization and use of new peptide substrates for assaying glycogen synthase kinase-3 (GSK-3) which are based on the sequence around the single GSK-3 phosphorylation site in the translation factor eIF2B. The new peptides offer important advantages over previous substrates, which were based on the sequence around the multiple GSK-3 phosphorylation sites in glycogen synthase (GS), for the assay of GSK-3 in cell extracts. In particular, decreases in GSK-3 activity following, e.g., insulin treatment, are partially or completely masked when the GS-based peptides are used but are readily measured using the new, eIF2B-based, peptides. The new peptides, unlike those based on GS, are therefore suitable for the assay of changes in GSK-3 activity in cell extracts without the need for prior immunoprecipitation or ion-exchange chromatography.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3 Cells
  • Adipocytes
  • Amino Acid Sequence
  • Animals
  • CHO Cells
  • Calcium-Calmodulin-Dependent Protein Kinases / analysis*
  • Calcium-Calmodulin-Dependent Protein Kinases / chemistry
  • Cell Extracts / chemistry
  • Cricetinae
  • Glycogen Synthase Kinase 3
  • Glycogen Synthase Kinases
  • Insulin / pharmacology
  • Mice
  • Molecular Sequence Data
  • Peptides / chemical synthesis*
  • Peptides / chemistry
  • Peptides / genetics
  • Phosphorylation

Substances

  • Cell Extracts
  • Insulin
  • Peptides
  • Glycogen Synthase Kinases
  • Calcium-Calmodulin-Dependent Protein Kinases
  • Glycogen Synthase Kinase 3

Associated data

  • GENBANK/U19516
  • GENBANK/U23028