1997 Fiscal Year Final Research Report Summary
Development of methods for detection of various protein kinases in sodium dodecyl sulfate-polyacrylamide gel
| Project/Area Number |
08557012
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Pathological medical chemistry
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| Research Institution | Asahikawa Medical College |
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Principal Investigator |
FUJISAWA Hitoshi Asahikawa Medical College Biochemistry Professor, 医学部, 教授 (10027039)
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| Co-Investigator(Kenkyū-buntansha) |
ISHIDA Atsuhiko Asahikawa Medical College Biochemistry Instructor, 医学部, 助手 (90212886)
KAMESHITA Isamu Asahikawa Medical College Biochemistry Assistant Professor, 医学部, 助教授 (60127941)
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| Project Period (FY) |
1996 – 1997
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| Keywords | Polyacrylamide gel electrophoresis / assay method / protein kinase / protein phosp hatase / synthetic oligopeptide / protein phosphorylation / protein dephosphorylation / signal transduction |
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| Research Abstract |
We had developed a sensitive method for detection of protein kinase activities in gels after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In this method, the in situ renaturation of proteins in gels after SDS-PAGE was applied to the detection of protein kinase either autophosphorylation or by the phosphorylation of protein substrates included in the gel. However, extensive use of this method was hampered by the need for a relatively large amounts of substrate proteins, and therefore the use of synthetic oligopeptides in place of proteins as substrates were tried. The oligopeptides which were linked to amino acid polymers such as poly-L-lysine through their amino-terminal cysteinyl residue by a heterobifunctional reagent were efficiently retained in the gel matrix and served as substrates for protein kinases. Another advantage of the use of synthetic oligopeptides is that the sequence of the oligopeptides can be designed to be suited for studies of various protein kinases. Thus, various protein kinases in the crude tissue extracts were efficiently detected by various synthetic oligopeptides included in the gel.
A method for detection of protein phosphatase activities toward phosphorylated oligopeptides in SDS-PAGE was also developed. With the use of a synthetic peptide corresponding to the autophosphorylation site of calmodulin-dependent protein kinase II (CaM-kinase II) which was conjugated to poly-L-lysine and then phosphorylated with [gamma-^<-32>P]ATP by the action of CaM-kinase II,as a substrate, several new protein phosphatase were detected in crude rat brain extracts. One of them was purified and characterized. The results suggest that the purifed eazyme is a specialized protein phosphatase for the regulation of CaM-kinase II.
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Research Products
(38 results)
