Detection of protein phosphatase activities in sodium dodecyl sulfate-polyacrylamide gel using peptide substrates.
A method for detection of protein phosphatase activity toward phosphorylated oligopeptides in SDS-polyacrylamide gel was developed. A synthetic peptide (MHRQETVDC) corresponding to the autophosphorylation site of calmodulin-dependent protein kinase II (residue 281-289) was conjugated to poly-L-lysine and phosphorylated with [gamma-32P]ATP by the action of calmodulin-dependent protein kinase II, and the [32P]-phosphopeptide-polymer conjugate was included as a substrate for protein phosphatases in gels. When a crude extract from rat brain was electrophoresed on polyacrylamide gel containing the [32P]phosphopeptide conjugate, followed by treatment for in situ renaturation and autoradiography, three transparent bands corresponding to apparent molecular weights of 52,000, 58,000 and 74,000, resulting from the removal of the [32P]phosphate from the phosphopeptide conjugate included in the gel were observed, indicating the existence of at least three different phosphoprotein phosphatases catalyzing dephosphorylation of the phosphopeptide in the brain. Among the three, two bands corresponding to molecular weights of 52,000 and 58,000 were not clearly observed when other phosphopeptide-polymer conjugates such as C-syntide-2 and CAMKAKS peptide were included in gels, suggesting that site-specific protein phosphatases can be detected in crude tissue extracts by this in-gel protein phosphatase assay.
Kameshita
I
I
Department of Biochemistry, Asahikawa Medical College, Japan.
Ishida
A
A
Okuno
S
S
Fujisawa
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002531:Cerebellum / Q000201:enzymology / Q000254:growth & development; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004789:Enzyme Activation; D018507:Gene Expression Regulation, Developmental; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000032:analysis* / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013379:Substrate Specificity; D014018:Tissue Distribution; D014020:Tissue Extracts / Q000737:chemistry
D010455:Peptides; D014020:Tissue Extracts; D012967:Sodium Dodecyl Sulfate; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases