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

用蛋白激酶抑制剂亲和层析法成功地从兔脑中分离纯化了蛋白激酶C，并从鸡肌球蛋白轻链和人血小板中分离纯化了肌球蛋白轻链蛋白。用纯化的C蛋白进行氨基酸序列测定。对兔三种不同类型的蛋白激酶C(α、β和GAM)的互补DNA克隆进行了鉴定和测序，然后用羟基磷灰石柱层析将其分为三种亚型。我们获得了三种类型的蛋白激酶C单抗，它们可以选择性地与羟基磷灰石柱层析分离的同工酶相互作用，即I、II和III型蛋白激酶。为阐明蛋白激酶C在血管平滑肌收缩中的作用，本实验观察了外源性蛋白激酶C对兔肠系膜动脉平滑肌标本的作用。已阐明肌球蛋白轻链蛋白激酶C的磷酸化在血管平滑肌收缩中起抑制作用。MLCK被A激酶(2摩尔磷酸盐/MLCK摩尔)磷酸化。甲状旁腺素被认为是A蛋白磷酸化的一个部位是钙调蛋白结合部位。利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，蛋白激酶抑制剂-亲和层析可用于研究纯化的蛋白激酶的生理功能，并研究各种蛋白激酶之间的相互关系。

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.