Chimaeras reveal the role of the catalytic core in the activation of the plasma membrane Ca2+ pump.
Chimaeras reveal the role of the catalytic core in the activation of the plasma membrane Ca2+ pump.
复制标题
Chimaeras 揭示了催化核心在质膜 Ca2 泵激活中的作用。
DOI：
10.1042/0264-6021:3560241
复制
发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Penniston,JT
中科院分区：
文献类型：
--
作者：
Ba-Thein,W;Caride,AJ;Enyedi,A;Pászty,K;Croy,CL;Filoteo,AG;Penniston,JT
质膜钙泵的亚型2b与普遍存在的亚型4b的不同之处在于：(A)在没有钙调蛋白的情况下，基础活性更高；(B)对钙调蛋白的亲和力更高；(C)在钙调蛋白的存在下，对钙的亲和力更高[Elwess，Filoteo，Enyedi和Penniston(1997)J.Biol]。化学272,17981-17986]。为了研究分子的哪些部分决定了这些动力学差异，我们制作了四个嵌合体结构，其中部分异构体2b被嫁接到异构体4b中：嵌合体I只包含异构体2b的C-末端调节区；嵌合体II包含异构体2b的N端部分，包括两个细胞质环；嵌合体III包含从N端到第一个(小)细胞质环结束之后的异构体2b序列；嵌合体IV只包含第二个(大的)细胞质环。令人惊讶的是，嵌合体I在没有钙调蛋白的情况下表现出低的基础活性，并且与异构体2b不同，对钙调蛋白的亲和力很低。相反，包含这两个环的嵌合体显示出高的基础活性，并且钙激活曲线(无论在没有和存在钙调蛋白的情况下)与异构体2b的曲线相似。测定了钙调素的激活速率和去除钙调蛋白的失活速率，并根据这两个速率常数的比值计算了钙调素的表观Kd。亲和力顺序为：2B=II>；4b=IV>；III=I。从这些结果可以清楚地看出，与异构体2b最相似的结构是嵌合体II。这表明，要获得与异构体2b性质相似的酶，需要两个细胞质环。
Isoform 2b of the plasma membrane calcium pump differs from the ubiquitous isoform 4b in the following: (a) higher basal activity in the absence of calmodulin; (b) higher affinity for calmodulin; and (c) higher affinity for Ca2+in the presence of calmodulin [Elwess, Filoteo, Enyedi and Penniston (1997) J. Biol. Chem.272, 17981–17986]. To investigate which parts of the molecule determine these kinetic differences, we made four chimaeric constructs in which portions of isoform 2b were grafted into isoform 4b: chimaera I contains only the C-terminal regulatory region of isoform 2b; chimaera II contains the N-terminal moiety of isoform 2b, including both cytoplasmic loops; chimaera III contains the sequence of isoform 2b starting from the N-terminus to after the end of the first (small) cytoplasmic loop; and chimaera IV contains only the second (large) cytoplasmic loop. Surprisingly, chimaera I showed low basal activity in the absence of calmodulin and low affinity for calmodulin, unlike isoform 2b. In contrast, the chimaera containing both loops showed high basal activity, and Ca2+activation curves (both in the absence and in the presence of calmodulin) similar to those of isoform 2b. The rates of activation by calmodulin and of inactivation by calmodulin removal were measured, and the apparentKdfor calmodulin was calculated from the ratio between these rate constants. The order of affinity was: 2b = II>4b = IV>III = I. From these results it is clear that the construct that most closely resembles isoform 2b is chimaera II. This shows that, in order to obtain an enzyme with properties similar to those of isoform 2b, both cytoplasmic loops are needed.