Protein phosphatase assay using a modification of the P81 paper protein kinase assay procedure.
Synthetic peptides have been used to define specificity determinants and to distinguish reactivities of numerous protein kinases and phosphoprotein phosphatases. Direct analysis of peptide phosphorylation is most often determined using P81 phosphocellulose paper to separate modified peptide and unreacted [gamma-32P]ATP; however phosphopeptide dephosphorylation is usually determined by extraction and quantitation of phosphomolybdate complexes or ion exchange chromatography. We describe here the adaptation of the rapid, direct P81 paper protein kinase assay for the determination of phosphopeptide dephosphorylation. The S6-21 peptide (AKRRRLSSLRASTSKSESSQK), which is derived from the multiphosphorylated carboxyl terminal domain of the S6 ribosomal protein, was phosphorylated by a human placenta S6 kinase and dephosphorylation by purified phosphoprotein phosphatase type 1 in the presence of a variety of buffers, and inhibitors/activators was determined using the new assay. Results comparable to those obtained with the ion-exchange chromatography were obtained, and the assay was significantly less expensive, more rapid, and more accurate than methods previously used to quantitate phosphopeptide dephosphorylation.
Abukhalaf
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Division of Biochemistry, University of North Texas, Denton 76203.
Masaracchia
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RA
D000595:Amino Acid Sequence; D000818:Animals; D002411:Cation Exchange Resins; D002482:Cellulose / Q000031:analogs & derivatives; D002623:Chemistry Techniques, Analytical / Q000379:methods; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010209:Paper; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000032:analysis*; D010750:Phosphoproteins / Q000032:analysis; D010766:Phosphorylation; D011494:Protein Kinases / Q000032:analysis*; D011506:Proteins / Q000032:analysis / Q000378:metabolism; D011817:Rabbits; D012269:Ribosomal Proteins / Q000032:analysis / Q000378:metabolism
D002411:Cation Exchange Resins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010750:Phosphoproteins; D011506:Proteins; D012269:Ribosomal Proteins; C081590:S6-21 peptide; D002482:Cellulose; C008881:phosphocellulose; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases