淀粉样蛋白Aβ_(1-42)与α_1-抗糜蛋白酶反应的体外研究  

In vitro study of interaction between Aβ_(1-42) peptide and α_1-antichymotrypsin

作　　者：孙永馨[1] 贾建平[1] Sabina Janciauskiene 

机构地区：[1]首都医科大学宣武医院,北京100053 [2]Department of Medicne,Wallenberg Laboratory,Malm University Hospital

出　　处：《中国神经精神疾病杂志》2010年第4期220-224,共5页Chinese Journal of Nervous and Mental Diseases

基　　金：国家自然科学基金面上项目(编号:30600656);北京市科技新星计划B类资助项目(编号:2004B12)

摘　　要：目的研究淀粉样蛋白Aβ1-42与α1-抗糜蛋白酶(α1-antichymotrypsin,ACT)在体外动态反应的性质,为阿尔茨海默病(Alzheimer's disease,AD)发病机制的研究探索新的领域。方法Aβ1-42、ACT以10:1的摩尔比溶解于Tris缓冲液中,37℃下孵育7h形成复合物。SDS-PAGE及糜蛋白酶(chymotrypsin,CHY)干扰实验探索Aβ1-42/ACT复合物的性质;采用ACT蛋白酶抑制剂活性的化学计量法研究和荧光标记Aβ1-42的解离追踪计算Aβ1-42与ACT的结合速率和解离速率;定量分析Aβ1-42浓度对ACT活性的影响。结果Aβ1-42与ACT反应7h形成的复合物在SDS-PAGE电泳条件下不稳定,ACT仍具有蛋白酶抑制剂活性。Aβ1-42与ACT结合后,后者的蛋白酶抑制剂活性减弱约10倍(SI=1.1vsSI=10.5),并且在一定范围内ACT的活性随Aβ1-42浓度的增高而减弱。Aβ1-42/ACT的结合速率常数为1.7(M.s)-1,解离速率常数为1.4×10-5s-1,即37℃、PH值7.4条件下Aβ1-42与ACT的结合速率远远高于解离速率。结论体外定量实验证实Aβ1-42、ACT之间的动态反应可形成稳定复合物并最终使ACT丧失蛋白酶抑制剂活性,提示ACT在AD的发病中可能发挥着与Aβ1-42相关的重要作用。Objective To characterize the kinetic reaction between Aβ1-42 and α1-antichymotrypsin （ACT） in vitro,and to explore new pathways for the pathogenic study of Alzheimer＇s disease （AD）.Methods Aβ1-42 and ACT were dissolved in Tris buffer solution at a molar ratio of 10：1 to form Aβ1-42/ACT complex under 37℃ for 7 h.The kinetics of formation and breakdown of this complex were determined from SDS-PAGE by adding chymotrypsin （CHY）.Stoichiometry of inhibition （SI） test of ACT and fluorescence measurements of fluorescein-labelled Aβ1-42 （fAβ1-42） were used for calculation of Kon and Koff between Aβ1-42 and ACT.Protease inhibitor activity of ACT was also studied under various Aβ1-42 concentrations.Results ACT and Aβ1-42 formed SDS-labile complex after 7 h incubation,ACT remained active for protease inhibition.After interaction with Aβ1-42,the protease inhibitor activity of ACT was diminished about ten times compared with ACT alone （SI=1.1 vs SI=10.5）,and ACT activity was dependent on Aβ1-42 concentration.The calculated Kon and Koff values of Aβ1-42 and ACT interaction were 1.7 （M·s）^-1 and 1.4×10^-5 s^-1 respectively,indicating that the two molecules combines with each other much faster than their dissociation at 37℃,pH 7.4.Conclusions The present quantitative analysis confirmed a complex formation between Aβ1-42 and ACT in vitro,as a result,the protease activity of ACT was lost.These results support an important role of ACT in Alzheimer＇s pathogenesis involving Aβ1-42.

关 键 词：淀粉样蛋白Aβ1-42 阿尔茨海默病 Α1-抗糜蛋白酶 蛋白酶 

分 类 号：R741.02[医药卫生—神经病学与精神病学] R749.16[医药卫生—临床医学]