Filipek A, Wojda U, Leśniak W
Department of Muscle Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland.
Int J Biochem Cell Biol. 1995 Nov;27(11):1123-31. doi: 10.1016/1357-2725(95)00096-8.
The structural properties of calcyclin protein are quite well characterized but its function remains obscure. To help elucidate the biological role of calcyclin we have performed the in vitro studies of the Ca(2+)-dependent interaction of Ehrlich ascites tumor cells calcyclin and its cyanogen bromide fragments with two potential calcyclin targets: annexin II and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The binding of annexin II, evidenced by the reaction with 125I-calcyclin, was found to be very weak and occurred only for intact calcyclin. On the other hand the interaction between calcyclin and GAPDH was of high affinity and could be assigned to the N-terminal region of calcyclin. Intact calcyclin and its N-terminal fragment bound to GAPDH in the gel overlay and affinity chromatography assay. When examined in the presence of a crosslinking agent the interaction resulted in the formation of 46K covalent adduct between calcyclin monomer and GAPDH subunit. Fluorescence of 5-iodoacetamido-fluorescein-labelled calcyclin was efficiently quenched by GAPDH in the presence of Ca2+. Titration experiments revealed the stoichiometry of one calcyclin monomer binding to each of GAPDH subunits with a binding constant of 10(8) M-1. The results of this work suggest that the binding between calcyclin and GAPDH may have bearing on calcyclin function.
钙结合蛋白的结构特性已得到相当充分的表征，但其功能仍不清楚。为了帮助阐明钙结合蛋白的生物学作用，我们进行了体外研究，研究了艾氏腹水瘤细胞钙结合蛋白及其溴化氰片段与两种潜在的钙结合蛋白靶点：膜联蛋白II和甘油醛-3-磷酸脱氢酶（GAPDH）之间的钙依赖性相互作用。通过与125I-钙结合蛋白的反应证明，膜联蛋白II的结合非常弱，且仅发生在完整的钙结合蛋白上。另一方面，钙结合蛋白与GAPDH之间的相互作用具有高亲和力，并且可以归因于钙结合蛋白的N端区域。完整的钙结合蛋白及其N端片段在凝胶覆盖和亲和色谱分析中与GAPDH结合。当在交联剂存在下进行检测时，相互作用导致钙结合蛋白单体与GAPDH亚基之间形成46K共价加合物。在Ca2+存在下，GAPDH有效地淬灭了5-碘乙酰胺-荧光素标记的钙结合蛋白的荧光。滴定实验揭示了一个钙结合蛋白单体与每个GAPDH亚基结合的化学计量比，结合常数为10(8) M-1。这项工作的结果表明，钙结合蛋白与GAPDH之间的结合可能与钙结合蛋白功能有关。
