Cyclic AMP-dependent protein kinases of Paramecium. II. Catalytic and regulatory properties of type II kinase from cilia.
Cyclic AMP-dependent protein kinases of Paramecium. II. Catalytic and regulatory properties of type II kinase from cilia.
复制标题
草履虫的环状 AMP 依赖性蛋白激酶。
DOI：
10.1016/0167-4889(89)90191-2
复制
发表时间：
1989
期刊：
影响因子：
--
通讯作者：
Nelson,DL
中科院分区：
文献类型：
--
作者：
Mason,PA;Nelson,DL
草履虫纤毛的II型cAMP依赖的蛋白激酶(cAMP-PK-II)，不含cAMP-PK(cAMP-PK-I)和cGMP依赖的蛋白激酶(cGMP-PK)，能磷酸化几种碱性蛋白和一个含丝氨酸的七肽(Kemptie)。该酶可被蛋白激酶抑制剂(沃尔什抑制剂)部分抑制，但仅在相对较高的抑制剂浓度下被抑制。CAMP-PK-II的半峰激活出现在cAMP 15~25 nM。测试了几个cAMP类似物结合和激活酶的能力。8-bromo-cGMP是草履虫cGMP-PK的有效激活剂，而对草履虫cAMP-PK-II的激活剂较差。磷酸化分析缓冲液对cAMP-PK-II的激活有影响。与MOPS-Tris缓冲液相比，磷酸盐缓冲液增加了cAMP的激活，但降低了总活性。当测定缓冲液的pH较高时，该激酶不依赖于cAMP。在没有cAMP的情况下，cAMP-PK-II与组蛋白预先孵育也能激活该酶。CAMP-PK-II结合cAMP的Kd值为23 nM，结合cAMP的释放具有双时相过程，提示有两个不相同的结合部位。这种有纤毛的原生动物的cAMP-PK的性质似乎与脊椎动物的非常相似。
The type II cAMP-dependent protein kinase (cAMP-PK-II) from cilia ofParamecium, purified free of type I cAMP-PK (cAMP-PK-I) and of cGMP-dependent protein kinase (cGMP-PK), phosphorylated several basic proteins and a heptapeptide containing serine (Kemptide). The enzyme was partially inhibited by the protein kinase inhibitor (Walsh inhibitor), but only at relatively high inhibitor concentrations. Half-maximal activation of cAMP-PK-II occurred at 15–25 nM cAMP. Several cAMP analogs were tested for ability to bind and activate the enzyme. 8-bromo-cGMP, a potent activator ofParameciumcGMP-PK, was a poor activator ofParameciumcAMP-PK-II. Activation of cAMP-PK-II was influenced by the phosphorylation assay buffer. Phosphate buffers provided increased activation by cAMP but decreased total activity relative to that measured in Mops-Tris buffer. The kinase was cAMP-independent when the pH of the assay buffer was high. Preincubation of cAMP-PK-II with histones also activated the enzyme in the absence of cAMP. The cAMP-PK-II bound cAMP with aKdof 23 nM, and bound cAMP was released with a biphasic time course, suggesting two non-identical binding sites. The properties of the cAMP-PK of this ciliated protozoan appear to be closely similar to those of vertebrates.