Kincaid R L, Vaughan M
Proc Natl Acad Sci U S A. 1986 Mar;83(5):1193-7. doi: 10.1073/pnas.83.5.1193.
The mechanism of Ca2+-dependent protein-protein interaction and enzyme activation by calmodulin was investigated with the phosphoprotein phosphatase, calcineurin. Dimethylaminonaphthalene (dansyl)-calmodulin, a fluorescent derivative used to monitor complex formation, produced similar maximal activation (10- to 12-fold) with a Ca2+ dependence (Ka = 17 microM) identical to that of native calmodulin. The Ca2+-dependent increase in fluorescence intensity of dansyl-calmodulin was enhanced 100-150% by calcineurin, indicating complex formation; the concentration of Ca2+ required for a half-maximal increase in fluorescence was the same (K1/2 approximately equal to 7 microM) with and without calcineurin. Since the Ca2+ concentration required for activation appeared to differ from that necessary for protein-protein interaction, a method was devised to measure both the formation of complexes between dansyl-calmodulin and calcineurin and enzyme activity in the same samples. Direct comparison of interaction (measured by polarization of fluorescence) and enzyme activity demonstrated different Ca2+ requirements for the two events. Whereas dansyl-calmodulin-calcineurin interaction, measured in the presence of phosphoprotein substrate, exhibited very little cooperativity (Hill coefficient = 1.2, Ca2+ concentration required for the half-maximal increase in fluorescence, K1/2, approximately equal to 6 microM), phosphatase activation was highly cooperative (Hill coefficient = 3.5) and required 3 times higher Ca2+ concentration for half-maximal stimulation. Equivalent results were obtained with p-nitrophenyl phosphate as substrate. These data are consistent with a sequential mechanism for interaction and activation wherein filling of perhaps two Ca2+ sites permits calmodulin interaction with the phosphatase; this complex is inactive, requiring further binding of Ca2+ for activation. Such a scheme would provide a sensitive switch for control of enzyme activity within a narrow range of free Ca2+ concentration.
利用磷酸蛋白磷酸酶钙调神经磷酸酶研究了钙调蛋白依赖Ca2+的蛋白质-蛋白质相互作用及酶激活机制。二甲基氨基萘（丹磺酰）-钙调蛋白是一种用于监测复合物形成的荧光衍生物，它能产生与天然钙调蛋白相似的最大激活作用（10至12倍），且对Ca2+的依赖性（Ka = 17 microM）相同。钙调神经磷酸酶使丹磺酰-钙调蛋白荧光强度的Ca2+依赖性增加提高了100 - 150%，表明形成了复合物；在有和没有钙调神经磷酸酶的情况下，荧光强度半最大增加所需的Ca2+浓度相同（K1/2约等于7 microM）。由于激活所需的Ca2+浓度似乎与蛋白质-蛋白质相互作用所需的浓度不同，因此设计了一种方法来测量同一样品中丹磺酰-钙调蛋白与钙调神经磷酸酶之间复合物的形成以及酶活性。对相互作用（通过荧光偏振测量）和酶活性的直接比较表明，这两个事件对Ca2+的需求不同。在磷酸蛋白底物存在下测量的丹磺酰-钙调蛋白-钙调神经磷酸酶相互作用表现出很小的协同性（希尔系数 = 1.2，荧光强度半最大增加所需的Ca2+浓度，K1/2，约等于6 microM），而磷酸酶激活具有高度协同性（希尔系数 = 3.5），半最大刺激所需的Ca2+浓度高3倍。以对硝基苯磷酸为底物也获得了等效结果。这些数据与一种相互作用和激活的顺序机制一致，其中可能两个Ca2+位点的填充允许钙调蛋白与磷酸酶相互作用；这种复合物无活性，需要进一步结合Ca2+才能激活。这样的方案将在游离Ca2+浓度的窄范围内为酶活性的控制提供一个灵敏的开关。
