A new peptide conjugate as a highly specific substrate for MAP kinase.
A synthetic peptide (APRTPGGRC) cross-linked to poly-L-lysine through a carboxy-terminal cysteinyl residue was found to be a highly specific substrate for mitogen-activated protein (MAP) kinases. This peptide conjugate exhibited a much lower Km value (74 microM) than the free peptide substrate (APRTPGGRR, Km > 1 mM) previously used as a specific substrate for MAP kinases. Unlike myelin basic protein, which has been often used as a substrate for MAP kinases, this conjugate did not serve as substrate for cAMP-dependent protein kinase, protein kinase C, or multifunctional calmodulin-dependent protein kinases. Using the peptide conjugate as a substrate, MAP kinase activities in crude cell extracts were directly determined by in vitro assay and specifically detected by in-gel assay.
Kameshita
I
I
Department of Biochemistry, Asahikawa Medical College, Hokkaido.
Ishida
A
A
Fujisawa
H
H
D000818:Animals; D001665:Binding Sites; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000187:drug effects / Q000378:metabolism*; D002457:Cell Extracts; D004586:Electrophoresis / Q000379:methods; D004676:Myelin Basic Protein / Q000378:metabolism*; D009414:Nerve Growth Factors / Q000494:pharmacology; D016716:PC12 Cells / Q000187:drug effects / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
D002457:Cell Extracts; D004676:Myelin Basic Protein; D009414:Nerve Growth Factors; D010446:Peptide Fragments; D010455:Peptides; D011493:Protein Kinase C; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C517686:Camk4 protein, rat