Reiffen F U, Gratzl M
Biochemistry. 1986 Jul 29;25(15):4402-6. doi: 10.1021/bi00363a034.
Recently we found that Ca2+ within chromaffin vesicles is largely bound [Bulenda, D., & Gratzl, M. (1985) Biochemistry 24, 7760-7765]. In order to explore the nature of these bonds, we analyzed the binding of Ca2+ to the vesicle matrix proteins as well as to ATP, the main nucleotide present in these vesicles. The dissociation constant at pH 7 is 50 microM (number of binding sites, n = 180 nmol/mg of protein) for Ca2+-protein bonds and 15 microM (n = 0.8 mumol/mumol) for Ca2+-ATP bonds. When the pH is decreased to more physiological values (pH 6), the number of binding sites remains the same. However, the affinity of Ca2+ for the proteins decreases much less than its affinity for ATP (dissociation constant of 90 vs. 70 microM). At pH 6 monovalent cations (30-50 mM) as well as Mg2+ (0.1-0.5 mM), which are also present within chromaffin vesicles, do not affect the number of binding sites for Ca2+ but cause a decrease in the affinity of Ca2+ for both proteins and ATP. For Ca2+ binding to ATP in the presence of 0.5 mM Mg2+ we found a dissociation constant of 340 microM and after addition of 35 mM K+ a dissociation constant of 170 microM. Ca2+ binding to the chromaffin vesicle matrix proteins in the presence of 0.5 mM Mg2+ is characterized by a Kd of 240 microM and after addition of 15 mM Na+ by a Kd of 340 microM.(ABSTRACT TRUNCATED AT 250 WORDS)
最近我们发现，嗜铬小泡内的Ca2+大部分是结合状态的[布伦达，D.，&格拉茨尔，M.（1985年）《生物化学》24卷，7760 - 7765页]。为了探究这些结合键的性质，我们分析了Ca2+与小泡基质蛋白以及与ATP（这些小泡中主要的核苷酸）的结合情况。在pH 7时，Ca2+与蛋白结合键的解离常数为50微摩尔（结合位点数，n = 180纳摩尔/毫克蛋白），Ca2+与ATP结合键的解离常数为15微摩尔（n = 0.8微摩尔/微摩尔）。当pH降至更接近生理值（pH 6）时，结合位点数保持不变。然而，Ca2+对蛋白的亲和力下降幅度远小于其对ATP的亲和力（解离常数分别为90微摩尔和70微摩尔）。在pH 6时，嗜铬小泡内也存在的单价阳离子（30 - 50毫摩尔）以及Mg2+（0.1 - 0.5毫摩尔），不会影响Ca2+的结合位点数，但会导致Ca2+对蛋白和ATP的亲和力下降。对于在0.5毫摩尔Mg2+存在下Ca2+与ATP的结合，我们发现解离常数为340微摩尔，加入35毫摩尔K+后解离常数为170微摩尔。在0.5毫摩尔Mg2+存在下，Ca2+与嗜铬小泡基质蛋白的结合特征是解离常数为240微摩尔，加入15毫摩尔Na+后解离常数为340微摩尔。（摘要截选至250字）
