1988 Fiscal Year Final Research Report Summary
Development of analytical method of molecular function in protein kinases by using newly synthesized protein kinase inhibitor - affinity chromatography
| Project/Area Number |
62880018
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| Research Category |
Grant-in-Aid for Developmental Scientific Research
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| Allocation Type | Single-year Grants |
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| Research Field |
物質生物化学
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| Research Institution | Nagoya University School of Medicine (1988)
Mie University (1987) |
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Principal Investigator |
HIDAKA Hiroyoshi Nagoya University School of Medicine, Professor, 医学部, 教授 (80100171)
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| Co-Investigator(Kenkyū-buntansha) |
中 充子 三重大学, 医学部, 助手 (10093139)
田中 利男 三重大学, 医学部, 教授 (00135443)
HAGIWARA Masatoshi Nagoya University School of Medicine, Staff, 医学部, 助手 (10208423)
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| Project Period (FY) |
1987 – 1988
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| Keywords | Myosin light chain kinase / ML-9 / Thyroxine / Affinity chromatography / Protein kinase C |
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| Research Abstract |
We succeed the purification of protein kinase C from rabbit brain and myosin light chain kinase (MLCK) from chicken gizzard and human platelet by using protein kinase inhibitor-affinity chromatography. The purified C kinase was used to determine amino acid sequence. Rabbit complementary DNA clones cording for three disinct types of protein kinase C, named alpha,beta and gam, have been identified and sequenced.Then, homogenous C-kinase were classified into three subtypes, using hydroxylapatite column chromatography. We obtained three types of protein kinase C monoclonal antibodies which selectively interact with hydroxyapatite column chromatographically resolved isozymes, type i, ii and iii of protein kinase. To clarify the role of protein kinase C in vascular smooth muscle contraction, the effects of exogenous protein kinase C was investigated on skinned smooth muscle preparations of the rabbit mesenteric artery. It was clarified that protein kinase C phosphorylation of myosin light chain play inhibit role in contraction of vascular smooth muscle. MLCK is phosphorylated by A kinase (2 mol of phosphate/mol of MLCK). It was recognized by using thyroxine that one site of phosphorylation by A kinase is calmodulin binding site. We clarified interrelationship among each protein kinase in vascular smooth muscle contraction mechanism using protein kinase inhibitor and affinity purified protein kinase. These evidence suggested that protein kinase inhibitor-affinity chromatography can be used to investigate the physiological function of purified protein kinase and to study interrelation among each protein kinase.
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