钙调蛋白片段的激动剂和拮抗剂特性。

Agonist and antagonist properties of calmodulin fragments.

作者信息

Newton D L, Oldewurtel M D, Krinks M H, Shiloach J, Klee C B

出版信息

J Biol Chem. 1984 Apr 10;259(7):4419-26.

Abstract

Limited proteolysis of calmodulin with trypsin in the presence of ethylene glycol bis(beta-aminoethyl ether)-N, N,N',N'-tetracetic acid (EGTA) or Ca2+ was performed according to a modification of the method of Drabikowski et al. (Drabikowski, W., Kuznicki, J., and Grabarek, Z. (1977) Biochim. Biophys. Acta 485, 124-133). The resulting peptides were purified by reverse-phase high performance liquid chromatography. Tryptic digests in EGTA yielded peptides 1-106, 1-90, and 107-148 with yields of 9, 47, and 61%, respectively. The digests performed with Ca2+ yielded peptides 1-77 and 78-148 in 35 and 45% yield. Analysis by high performance liquid chromatography indicated that the purified fragments contained less than 0.1% contamination by calmodulin, thus allowing a definitive study of the ability of these fragments to activate, or interact with, calmodulin-regulated enzymes and anti-calmodulin drugs. Each of the fragments, except 107-148, bound to a phenothiazine affinity column in a Ca2+-dependent manner. Thus, calmodulin contains two interaction sites for phenothiazines: one on the NH2-terminal half (fragment 1-77) and one on the COOH-terminal half (fragment 78-148). None of the fragments activates the protein phosphatase, calcineurin, or prevents its stimulation by calmodulin, nor does any of the fragments stimulate Ca2+-dependent cAMP phosphodiesterase. A single cleavage in the middle of the calmodulin molecule results in the rapid dissociation of the two resultant fragments and a loss of ability to activate cAMP phosphodiesterase. One fragment, 78-148, interacts with phosphodiesterase and prevents its activation by calmodulin (Ki: 1.5 +/- 0.4 X 10(-6) M). The same fragment, 78-148, can fully activate phosphorylase kinase but with a lower affinity than calmodulin (Kuznicki, J., Grabarek, Z., Brzeska, H., Drabikowski, W., and Cohen, P. (1981) FEBS Lett. 130, 141-145). Thus, peptide 78-148 behaves as a calmodulin agonist or antagonist or as neither, depending on the enzyme under study.

摘要

按照Drabikowski等人方法的改进（Drabikowski, W., Kuznicki, J., and Grabarek, Z. (1977) Biochim. Biophys. Acta 485, 124 - 133），在乙二醇双（β - 氨基乙醚）-N,N,N',N'-四乙酸（EGTA）或Ca²⁺存在的情况下，用胰蛋白酶对钙调蛋白进行有限的蛋白水解。所得肽段通过反相高效液相色谱进行纯化。在EGTA中进行的胰蛋白酶消化产生了肽段1 - 106、1 - 90和107 - 148，产率分别为9%、47%和61%。在Ca²⁺存在下进行的消化产生了产率分别为35%和45%的肽段1 - 77和78 - 148。高效液相色谱分析表明，纯化的片段中钙调蛋白的污染小于0.1%，从而能够对这些片段激活钙调蛋白调节酶或与抗钙调蛋白药物相互作用的能力进行确定性研究。除107 - 148外，每个片段都以Ca²⁺依赖的方式与吩噻嗪亲和柱结合。因此，钙调蛋白含有两个与吩噻嗪相互作用的位点：一个在NH₂末端的一半（片段1 - 77），另一个在COOH末端的一半（片段78 - 148）。这些片段均不能激活蛋白磷酸酶钙调神经磷酸酶，也不能阻止其被钙调蛋白刺激，并且没有任何一个片段能刺激Ca²⁺依赖的环磷酸腺苷磷酸二酯酶。钙调蛋白分子中部的一次切割会导致两个所得片段迅速解离，并丧失激活环磷酸腺苷磷酸二酯酶的能力。一个片段78 - 148与磷酸二酯酶相互作用，并阻止其被钙调蛋白激活（抑制常数Ki：1.5 ± 0.4×10⁻⁶ M）。同一个片段78 - 148可以完全激活磷酸化酶激酶，但亲和力低于钙调蛋白（Kuznicki, J., Grabarek, Z., Brzeska, H., Drabikowski, W., and Cohen, P. (1981) FEBS Lett. 130, 141 - 145）。因此，肽段78 - 148根据所研究的酶的不同，表现为钙调蛋白激动剂、拮抗剂或两者都不是。