Childs T J, Mak A S
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Biochem J. 1993 Dec 15;296 ( Pt 3)(Pt 3):745-51. doi: 10.1042/bj2960745.
A single 42 kDa isoform of mitogen-activated protein (MAP) kinase is expressed in both embryonic and adult chicken gizzard. The gizzard MAP kinase, which cross-reacts with anti-p44mpk antibody, has been purified from adult chicken gizzard and partially characterized. The purification protocol employs phenyl-Sepharose, polylysine-agarose, hydroxyapatite, Mono-Q and phenyl-Superose column chromatography. The purified enzyme phosphorylates myelin basic protein and gizzard high-molecular-mass (h-)caldesmon. Sea-star p44mpk and gizzard MAP kinase phosphorylate h-caldesmon at identical sites at the C-terminal domain, as revealed by tryptic-peptide mapping of the phosphorylated protein. Phosphorylation of h-caldesmon by gizzard MAP kinase abolishes its interaction with polymerized tubulin. The specific activity of the purified gizzard kinase toward myelin basic protein is similar to that of brain tau kinase, but is only a fraction of that of activated sea-star p44mpk. This suggests that, although a large amount of MAP kinase is present in the gizzard, only a small percentage of the enzyme is activated normally. Autophosphorylation of the gizzard kinase, at least in part on tyrosine residues, activates its kinase activity.
有丝分裂原激活蛋白（MAP）激酶的单一42 kDa亚型在胚胎期和成年鸡的砂囊中均有表达。与抗p44mpk抗体发生交叉反应的砂囊MAP激酶已从成年鸡砂囊中纯化出来，并进行了部分特性鉴定。纯化方案采用苯基琼脂糖凝胶、聚赖氨酸琼脂糖凝胶、羟基磷灰石、Mono-Q和苯基超琼脂糖柱色谱法。纯化后的酶可使髓鞘碱性蛋白和砂囊高分子量（h-）钙调蛋白磷酸化。胰蛋白酶肽图谱分析显示，海星p44mpk和砂囊MAP激酶在h-钙调蛋白C端结构域的相同位点使其磷酸化。砂囊MAP激酶使h-钙调蛋白磷酸化后，会消除其与聚合微管蛋白的相互作用。纯化后的砂囊激酶对髓鞘碱性蛋白的比活性与脑tau激酶相似，但仅为活化海星p44mpk比活性的一小部分。这表明，尽管砂囊中存在大量的MAP激酶，但通常只有一小部分酶被激活。砂囊激酶的自身磷酸化，至少部分是在酪氨酸残基上，可激活其激酶活性。
