Videen J S, Mezger M S, Chang Y M, O'Connor D T
Department of Medicine, University of California, San Diego 92161.
J Biol Chem. 1992 Feb 15;267(5):3066-73.
The soluble core of catecholamine storage vesicles in the adrenal medulla contains high concentrations of the cations calcium (20 mM) and catecholamine (600 mM). Do these cations interact with the abundant vesicle core anionic proteins, the chromogranins? We investigated the binding of calcium and norepinephrine (NE) to bovine adrenal chromogranins by equilibrium dialysis. Both calcium and NE were bound saturably by chromogranins, with low affinity (Kd values of 1.3 x 10(-4) M and 2.1 x 10(-3) M), but high capacity (17 and 32 mol of ligand/mol of chromogranin A). Both ligands bound maximally at a pH greater than 5.5 and were displaced by competing cations in a pattern (trivalent greater than divalent greater than monovalent) consistent with electrostatic components to the interactions. Binding of calcium and NE was not impaired by prior heat denaturation of the chromogranins, and chromogranin A was involved in both binding reactions. Calcium but not NE binding was enhanced by nonpolar solvents. Temperature dependence studies indicated that calcium binding to chromogranins was largely entropy-driven, while NE binding was driven by a significantly negative (favorable) change in enthalpy (5760 cal/mol), even in the face of an unfavorable entropy. Exposure of chromogranins to calcium or NE resulted in precipitation (aggregation) as analyzed by centrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NE was a more effective chromogranin precipitant than calcium, and in combination, the NE effect was antagonized by calcium. Precipitation of chromogranins by both calcium and NE was inhibited by NaCl at ionic strengths comparable with those of the ligands. These data suggest that chromogranins bind and are precipitated by calcium and NE at affinities compatible with their in situ concentrations, but that the interactions exhibit different thermodynamic driving forces. Furthermore, NE may trigger an enthalpy-driven conformational change in chromogranins, resulting in aggregation.
肾上腺髓质中儿茶酚胺储存囊泡的可溶性核心含有高浓度的阳离子钙（20 mM）和儿茶酚胺（600 mM）。这些阳离子是否与丰富的囊泡核心阴离子蛋白即嗜铬粒蛋白相互作用？我们通过平衡透析研究了钙和去甲肾上腺素（NE）与牛肾上腺嗜铬粒蛋白的结合。钙和NE均被嗜铬粒蛋白饱和结合，亲和力低（解离常数Kd值分别为1.3×10⁻⁴ M和2.1×10⁻³ M），但结合容量高（每摩尔嗜铬粒蛋白A可结合17和32摩尔配体）。两种配体在pH大于5.5时结合达到最大值，并被竞争性阳离子以一种与相互作用的静电成分一致的模式（三价大于二价大于一价）取代。嗜铬粒蛋白预先热变性并不损害钙和NE的结合，且嗜铬粒蛋白A参与了这两种结合反应。非极性溶剂可增强钙的结合，但不增强NE的结合。温度依赖性研究表明，钙与嗜铬粒蛋白的结合主要由熵驱动，而NE的结合则由显著的负（有利）焓变（5760卡/摩尔）驱动，即使面对不利的熵变。通过离心和十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分析，嗜铬粒蛋白暴露于钙或NE会导致沉淀（聚集）。NE是比钙更有效的嗜铬粒蛋白沉淀剂，并且两者共同作用时，钙会拮抗NE的作用。在与配体离子强度相当的情况下，NaCl可抑制钙和NE引起的嗜铬粒蛋白沉淀。这些数据表明，嗜铬粒蛋白以与其原位浓度相适应的亲和力与钙和NE结合并沉淀，但相互作用表现出不同的热力学驱动力。此外，NE可能引发嗜铬粒蛋白中焓驱动的构象变化，导致聚集。
