Mason P A, Nelson D L
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53706.
Biochim Biophys Acta. 1989 Jan 17;1010(1):116-21. doi: 10.1016/0167-4889(89)90191-2.
The type II cAMP-dependent protein kinase (cAMP-PK-II) from cilia of Paramecium, 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 of Paramecium cGMP-PK, was a poor activator of Paramecium cAMP-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 a Kd of 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.
从草履虫纤毛中纯化得到的II型环磷酸腺苷依赖性蛋白激酶（cAMP-PK-II），不含I型环磷酸腺苷依赖性蛋白激酶（cAMP-PK-I）和环磷酸鸟苷依赖性蛋白激酶（cGMP-PK），可使几种碱性蛋白和一种含丝氨酸的七肽（肯普肽）磷酸化。该酶受到蛋白激酶抑制剂（沃尔什抑制剂）的部分抑制，但仅在相对较高的抑制剂浓度下。cAMP-PK-II的半数最大激活浓度在15 - 25 nM环磷酸腺苷时出现。测试了几种环磷酸腺苷类似物结合和激活该酶的能力。8-溴环磷酸鸟苷是草履虫cGMP-PK的有效激活剂，但对草履虫cAMP-PK-II的激活作用较弱。cAMP-PK-II的激活受磷酸化测定缓冲液的影响。磷酸盐缓冲液可增加环磷酸腺苷的激活作用，但相对于在吗啉代丙磺酸-三羟甲基氨基甲烷缓冲液中测得的总活性有所降低。当测定缓冲液的pH值较高时，该激酶不依赖于环磷酸腺苷。cAMP-PK-II与组蛋白预孵育也可在无环磷酸腺苷的情况下激活该酶。cAMP-PK-II结合环磷酸腺苷的解离常数为23 nM，结合的环磷酸腺苷以双相时间进程释放，表明存在两个不同的结合位点。这种纤毛虫的环磷酸腺苷依赖性蛋白激酶的性质似乎与脊椎动物的非常相似。
