La3+诱导钙调蛋白与鼠脑组织钙调蛋白亲合蛋白的结合

应用固定化钙调蛋白(CaM)亲合色谱法、变性丙烯酰胺电泳(SDS-PAGE)和基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)等方法研究了La3+诱导CaM在大鼠脑匀浆液中的钙调蛋白亲合蛋白(CaMBP)谱以及CaM-CaMBP在模拟细胞环境下结合作用的La3+浓度依赖关系, 并与Ca2+的作用进行了比较. 实验结果表明, (1) La3+参与的CaMBP物种与Ca2+的基本相同. 鉴定了其中含量高且稳定出现的5种CaMBP分别是参与糖酵解反应的6-磷酸果糖激酶和3-磷酸甘油醛脱氢酶、细胞骨架类的微管蛋白和肌动蛋白以及应激反应相关的71000热休克同源蛋白, 表明稀土离子可能参与多种细胞过程; (2) La3+诱导CaM与5种CaMBP结合的浓度依赖曲线因CaMBP物种的不同而存在差别. 热休克同源蛋白、肌动蛋白或微管蛋白对La3+相对敏感, La3+在金属-CaM-CaMBP三元体系中与CaM的结合常数K与Ca2+的相近或稍高; 而6-磷酸果糖激酶和3-磷酸甘油醛脱氢酶体系对La3+的敏感性明显低于Ca2+. 其原因可能在于模拟细胞环境的复杂性以及CaM-CaMBP蛋白质相互作用对金属离子与CaM配位结合的调节.

La3+ Induced Binding of Calmodulin(CaM) to CaM-binding Proteins in Rat Brain Homogenate

Interactions of lanthanide ions with Ca2 /calmodulin(CaM)signal transduction is a crucial pathway for the biological effect of lanthanides.In the present work,the effect of La3 on binding of CaM to its binding proteins(CaMBP)was investigated by an immobilized CaM affinity chromatographic method in rat's brain homogenate.CaMBP binding to La3 -activated CaM gel were analyzed by SDS-PAGE and some CaMBP were identified by MALDI-TOF-MS.The concentration dependency on La3 for CaM-CaMBP binding was determined in a mimetic cellular media and compared with that of Ca2 .The experimental results showed that(1)the CaMBP species activated by La3 were primarily the same as those of Ca2 .Five selected CaMBP were identified to be 6-phosphofructokinase(PFK),glyceraldehyde-3-phosphate dehydrogenase(GAPDH)(enzymes in glycolysis),tubulin,actin(cytoskeleton proteins),and heat shock cognate 71000 protein(HSCP71,stress chaperone),indicating that La3 may interfere with multiple cellular processes through CaM.(2)The effective concentrations for La3 to induce binding of CaM to CaMBP depended on CaMBP species.Tubulin,actin and HSCP71 were observed to be sensitive to La3 revealed by the affinity constants K of La3 to CaM in the metal-CaM-CaMBP tertiary system,which are similar or higher than those of Ca2 .However,in the systems with PFK or GAPDH,La3 bound to CaM less tightly.We hypothesized that this may attribute to complexity of mimetic cellular media and/or the effects of CaM-CaMBP interaction on CaM-metal binding,on which further studies should be appropriate.The work may offer new insight to the mechanisms of the biological effects of lanthanides.