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Development of analytical method of molecular function in protein kinases by using newly synthesized protein kinase inhibitor - affinity chromatography

利用新合成的蛋白激酶抑制剂-亲和层析法开发蛋白激酶分子功能分析方法

基本信息

批准号：
62880018
负责人：
HIDAKA Hiroyoshi
金额：
$ 4.03万
依托单位：
Nagoya University School of Medicine (1988)Mie University (1987)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-62880018/
关键词：
Myosin light chain kinase ML-9 Thyroxine Affinity chromatography Protein kinase C ML-9 サイロキシン

项目摘要

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.

我们成功地用蛋白激酶亲和层析法从兔脑中分离纯化了蛋白激酶C，从鸡砂囊和人血小板中分离纯化了肌球蛋白轻链激酶（MLCK）。纯化的C激酶用于测定氨基酸序列。本研究对兔蛋白激酶C的三种不同类型（α、β和gam）的互补DNA克隆进行了鉴定和测序，并利用羟基磷灰石柱层析将同源的C激酶分为三种亚型。我们获得了三种类型的蛋白激酶C单克隆抗体，它们选择性地与羟基磷灰石柱层析分离的蛋白激酶I、II和III型同工酶相互作用。为了阐明蛋白激酶C在血管平滑肌收缩中的作用，研究了外源性蛋白激酶C对兔肠系膜动脉皮肤平滑肌制备物的影响。说明肌球蛋白轻链的蛋白激酶C磷酸化在血管平滑肌收缩中起抑制作用。MLCK被A激酶磷酸化（2 mol磷酸盐/mol MLCK）。用甲状腺素证明A激酶磷酸化的一个位点是钙调素结合位点。我们用蛋白激酶抑制剂和亲和纯化的蛋白激酶阐明了血管平滑肌收缩机制中各蛋白激酶之间的相互关系。这些证据表明，蛋白激酶亲和层析可用于研究纯化的蛋白激酶的生理功能和研究各蛋白激酶之间的相互关系。