Boldyreff B, Meggio F, Dobrowolska G, Pinna L A, Issinger O G
Institut für Humangenetik, Universität des Saarlandes, Homburg, Germany.
Biochim Biophys Acta. 1993 Apr 29;1173(1):32-8. doi: 10.1016/0167-4781(93)90239-a.
CKIIB, one of the CK-2 like enzymes which have been isolated from maize, has been shown to be a monomeric enzyme that cross-reacts with anti CK-2 alpha specific antibodies suggesting a possible relationship between the two proteins (Dobrowolska et al. (1992) Eur. J. Biochem. 204, 299-303). In order to support the immunological data also by biochemical and biophysical experiments the availability of a recombinant CK-2 alpha from maize was a prerequisite. A maize cDNA clone of maize CK-2 alpha was expressed in the bacterial strain BL21 (DE3). The recombinant protein was purified to homogeneity; its molecular mass on one-dimensional SDS PAGE was estimated to be 36.5 kDa. The calculated molecular mass according to the amino acid composition is 39,228 Da (332 amino acids). The recombinant maize CK-2 alpha (rmCK-2 alpha) exhibited mostly the same properties as the recombinant human CK-2 alpha (rhCK-2 alpha). In several respects it behaved differently from CKIIB, thus supporting the notion that either CKIIB is encoded by another gene or it undergoes extensive posttranscriptional and/or posttranslational alterations. Three observations in particular disprove any close relatedness between CKIIB and rmCK-2 alpha, namely: (a) the phosphorylation of calmodulin by CKIIB is dramatically stimulated by polylysine, whereas polylysine inhibits rather than stimulating the phosphorylation of calmodulin by rmCK-2 alpha (and by rhCK-2 alpha). (b) Addition of rhCK-2 beta has no significant influence on the stimulation of the calmodulin phosphorylation by CKIIB whereas in the case of rmCK-2 alpha and rhCK-2 alpha addition of rhCK-2 beta is required for optimal stimulation by polylysine. (c) CKIIB does not self-assemble with rhCK-2 beta to form a high molecular mass complex as it is demonstrated for rmCK-2 alpha.
CKIIB是从玉米中分离出的一种类CK - 2酶，已被证明是一种单体酶，它能与抗CK - 2α特异性抗体发生交叉反应，这表明这两种蛋白质之间可能存在某种关系（多布罗沃尔斯卡等人，（1992年）《欧洲生物化学杂志》204卷，299 - 303页）。为了通过生化和生物物理实验来支持免疫学数据，获得来自玉米的重组CK - 2α是一个先决条件。玉米CK - 2α的一个cDNA克隆在细菌菌株BL21（DE3）中表达。重组蛋白被纯化至同质；其在一维SDS - PAGE上的分子量估计为36.5 kDa。根据氨基酸组成计算的分子量为39228 Da（332个氨基酸）。重组玉米CK - 2α（rmCK - 2α）表现出与重组人CK - 2α（rhCK - 2α）基本相同的特性。在几个方面它与CKIIB表现不同，因此支持了这样一种观点，即要么CKIIB由另一个基因编码，要么它经历了广泛的转录后和/或翻译后改变。特别是有三个观察结果反驳了CKIIB与rmCK - 2α之间的任何密切关系，即：（a）CKIIB对钙调蛋白的磷酸化受到聚赖氨酸的显著刺激，而聚赖氨酸抑制而不是刺激rmCK - 2α（以及rhCK - 2α）对钙调蛋白的磷酸化。（b）添加rhCK - 2β对CKIIB刺激钙调蛋白磷酸化没有显著影响，而在rmCK - 2α和rhCK - 2α的情况下，添加rhCK - 2β是聚赖氨酸实现最佳刺激所必需的。（c）CKIIB不会像rmCK - 2α那样与rhCK - 2β自组装形成高分子量复合物。
