【摘要】 粉防已碱Tet是一种新的钙调蛋白（CaM）拮抗剂,它抑制CaM活化的Ca²⁺—Mg²⁺—ATP c。利用丹磺酰钙调蛋白（D—CaM）可方便地鉴测Tet与CaM的相互作用,直接证明了在CaM与Tet之间生成复合物CaM—Tet 我们制备的D—CaM,每个蛋白分子含1.3个丹磺酰基团,活化红细胞膜Ca²⁺—Mg²⁺—ATP c的生物活性与天然CaM基本相同。 当Ca²⁺与D—CaM结合时,丹磺酰基团环境疏水性增加,该荧光团产生较高的量子产率（增加1.6倍）,最大发射兰移（从512移兰至495nm）。存在Ca²⁺时,Tet使D—CaM的荧光强度进一步增加,谱线继续兰移,表明Ca²⁺及Tet诱导蛋白构象变化,改变了丹磺酰基团的微环境。 荧光滴定实验证明CaM与Tet结合的解离常数为1.8μM,两者的结合是绝对依赖Ca²⁺的。 药物Tet与CaM的结合可增强结合在CaM上的荧光探剂NPN的荧光,提示CaM不同疏水结合位之间存在变构相互作用。更多还原

【Abstract】 Tetrandrine is a new calmodulin(CaM)antagonist.It can inhibit activation of CaM-stimulated Ca2+ - Mg2+ - ATPase. Using a dansylcalmodulin (D-CaM) we provide a convenient and simple means of monitoring the interaction of tet with CaM.The direct evidence that shows the Ca2+ - dependent formation of complex CaM - Tet was obtained.Our D - CaM preparation contained approx.l.3mol of dansyl per mol of CaM and was basically indistinguishable from native CaM in their ability to activate the human erythrocyte Ca2+- Mg2+ - ATPase.Upon binding of Ca2+ to CaM the hydrophobicity of the environment of the dansyl group was increased producing a higher quantum yield of the fluorophore (by 1.6 fold)and blue shift in the λmax.of emission spectrum(from 512nm to 495 nm).When tet was added to D - CaM in the presence of Ca2+ the fluorescence intensity was further increased and blue shift in spectrum was more evident. These facts revealed that the Ca2+ and tet-induced conformational transition caused a change in the microsurrounding of the dansyl group.The fluorescence titration showed that CaM hound tet with a Kd value of 1.8/μM.This binding is absolutely Ca2+ - dependent.The association of tet with CaM can potentiate fluorescence probe(N - Phenyl - 1 - Naphthylamine)binding to CaM.These studies suggest that allosterie interaction occur among different hydrophobic binding sites on CaM. Abbreviations: CaM for calmodulin,Tet for tetrandrine.更多还原