Cooperstein S J, Watkins D T
Department of Anatomy, University of Connecticut Health Center, Farmington 06030, USA.
Arch Oral Biol. 1997 Aug;42(8):569-77. doi: 10.1016/s0003-9969(97)00050-2.
In studies designed to determine the mechanism by which Ca++ and calmodulin stimulate the fusion of parotid secretion granules with plasma membrane vesicles, the hypothesis tested was that Ca++ and calmodulin act by stimulating protein phosphorylation. It was earlier found that Ca++ and calmodulin, but neither alone, stimulated the phosphorylation of four secretion granule proteins with molecular masses of 64, 58, 55 and 31 kDa, and decreased the degree of phosphorylation of a 36-kDa protein. Further studies have shown that in the presence of an optimal concentration of calmodulin (2.4 microM), half-maximal activation of phosphorylation of the four proteins occurred at approx. 8 microM Ca++, and at a maximally effective Ca++ concentration (10(-4) M), half-maximal stimulation occurred at calmodulin concentrations between 0.13 and 1.1 microM for the different proteins. The studies now described also demonstrate that the need for calmodulin for stimulating the phosphorylation, but not the dephosphorylation, is specific; two other Ca(++)-binding proteins, parvalbumin and troponin, could not replace calmodulin in stimulating phosphorylation of the four secretion granule proteins, but either one could substitute for calmodulin in stimulating dephosphorylation of the 36-kDa protein. Additionally, the phosphorylated proteins appear to be located on the granule surface. When secretion granules were subjected to mild treatment with a concentration of trypsin that did not lyse the granules, the 31-, 36-, 55-, 58- and 64-kDa proteins were no longer observed. In the presence of optimal concentrations of Ca++ and calmodulin, a dose-dependent inhibition of the phosphorylation of the various proteins by two calmodulin antagonists, trifluoperazine and calmidazolium, was observed; 50% inhibition of phosphorylation of the different proteins was obtained at approx. 20-40 microM trifluoperazine and at about 2.5-3.0 microM calmidazolium. Inhibition of the dephosphorylation of the 36-kDa protein required greater concentrations of trifluoperazine and calmidazolium; 128 microM and 50 microM, respectively. These results are consistent with the hypothesis that the phosphorylation of one or more of the 31-, 55-, 58- and 64-kDa proteins, but not the dephosphorylation of the 36-kDa protein, may be involved in the action of Ca++ and calmodulin in secretion granule-plasma membrane fusion.
在旨在确定钙离子（Ca++）和钙调蛋白刺激腮腺分泌颗粒与质膜小泡融合机制的研究中，所检验的假设是Ca++和钙调蛋白通过刺激蛋白质磷酸化起作用。早期发现，Ca++和钙调蛋白（单独一种均无此作用）可刺激四种分子量分别为64、58、55和31 kDa的分泌颗粒蛋白的磷酸化，并降低一种36 kDa蛋白的磷酸化程度。进一步研究表明，在钙调蛋白最佳浓度（2.4 microM）存在的情况下，四种蛋白磷酸化的半最大激活发生在约8 microM Ca++时；在最大有效Ca++浓度（10^(-4) M）下，不同蛋白在钙调蛋白浓度介于0.13至1.1 microM之间时出现半最大刺激。现在所描述的研究还表明，钙调蛋白对刺激磷酸化（而非去磷酸化）的需求具有特异性；另外两种Ca(++)结合蛋白，小清蛋白和肌钙蛋白，在刺激四种分泌颗粒蛋白磷酸化时不能替代钙调蛋白，但二者中的任何一种在刺激36 kDa蛋白去磷酸化时均可替代钙调蛋白。此外，磷酸化蛋白似乎位于颗粒表面。当用不会使颗粒裂解的胰蛋白酶浓度对分泌颗粒进行温和处理时，不再观察到31、36、55、58和64 kDa的蛋白。在Ca++和钙调蛋白最佳浓度存在的情况下，观察到两种钙调蛋白拮抗剂三氟拉嗪和氯氮卓对各种蛋白磷酸化有剂量依赖性抑制；在约20 - 40 microM三氟拉嗪和约2.5 - 3.0 microM氯氮卓时，不同蛋白的磷酸化受到50%抑制。抑制36 kDa蛋白的去磷酸化需要更高浓度的三氟拉嗪和氯氮卓；分别为128 microM和50 microM。这些结果与以下假设一致，即31、55、58和64 kDa蛋白中的一种或多种的磷酸化（而非36 kDa蛋白的去磷酸化）可能参与Ca++和钙调蛋白在分泌颗粒 - 质膜融合中的作用。
