Gundersen R E, Nelson D L
J Biol Chem. 1987 Apr 5;262(10):4602-9.
The ciliated protozoan Paramecium tetraurelia contained two protein kinase activities that were dependent on Ca2+. We purified one of the enzymes to homogeneity by Ca2+-dependent affinity chromatography on phenyl-Sepharose and ion exchange chromatography. The purified enzyme contained polypeptides of 50 and 55 kDa, with the 50-kDa species predominant. From its Stokes radius (32 A) and sedimentation coefficient (3.9 S), we calculated a native molecular weight of 51,000, suggesting that the active form is a monomer. Its specific activity was 65-130 nmol X min-1 X mg-1 and the Km for ATP was 17-35 microM, depending on the exogenous substrate used. Kinase activity was completely dependent upon Ca2+; half-maximal activation occurred at approximately 1 microM free Ca2+ at pH 7.2. Phosphatidylserine and diacylglycerol did not stimulate activity, nor did the addition of purified Paramecium calmodulin. The enzyme phosphorylated casein and histones, forming primarily phosphoserine and phosphothreonine, respectively. It also catalyzed its own phosphorylation in a Ca2+-dependent reaction; the half-maximal rate of autophosphorylation occurred at approximately 1-1.5 microM free Ca2+, and both the 50- and 55-kDa species were autophosphorylated. After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renaturation in situ, the 50-kDa protein retained its Ca2+-dependent ability to phosphorylate casein, suggesting that Ca2+ interacts directly with this polypeptide. This was confirmed by direct binding studies; when the enzyme was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis transferred to nitrocellulose, and renatured, there was 45Ca2+-binding in situ to both the 50- and 55-kDa polypeptides. The Paramecium enzyme appears to be a new and unique type of Ca2+-dependent protein kinase.
纤毛虫四膜虫含有两种依赖钙离子的蛋白激酶活性。我们通过在苯基琼脂糖上进行钙离子依赖的亲和层析和离子交换层析，将其中一种酶纯化至同质。纯化后的酶含有50 kDa和55 kDa的多肽，其中50 kDa的种类占主导。根据其斯托克斯半径（32 Å）和沉降系数（3.9 S），我们计算出其天然分子量为51,000，表明活性形式是单体。其比活性为65 - 130 nmol·min⁻¹·mg⁻¹，ATP的Km值为17 - 35 μM，这取决于所使用的外源底物。激酶活性完全依赖于钙离子；在pH 7.2时，约1 μM游离钙离子下出现半数最大激活。磷脂酰丝氨酸和二酰基甘油不刺激活性，添加纯化的四膜虫钙调蛋白也无此作用。该酶使酪蛋白和组蛋白磷酸化，分别主要形成磷酸丝氨酸和磷酸苏氨酸。它还在钙离子依赖的反应中催化自身磷酸化；半数最大自磷酸化速率出现在约1 - 1.5 μM游离钙离子时，50 kDa和55 kDa的种类均被自磷酸化。经十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分离并原位复性后，50 kDa的蛋白质保留了其依赖钙离子使酪蛋白磷酸化的能力，表明钙离子直接与该多肽相互作用。直接结合研究证实了这一点；当酶经十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳转移至硝酸纤维素膜并复性后，50 kDa和55 kDa的多肽均有原位⁴⁵Ca²⁺结合。四膜虫的这种酶似乎是一种新型且独特的依赖钙离子的蛋白激酶。
