Fitzpatrick S, Waisman D M
Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
Mol Cell Biochem. 1996 Feb 23;155(2):121-30. doi: 10.1007/BF00229309.
Bovine chromaffin secretory granules were purified by isopycnic Metrizamide gradient centrifugation and their Ca2+ sequestration pathways were characterized. The rate of Ca2+ sequestration at 37 degrees C was first order, with a maximal uptake of 26.9 +/- 0.46 (mean +/- S.D., n = 3) nmol Ca2+/mg protein and a first order rate constant (k) of 0.046 +/- 0.002 min-1. At 4 degrees C the rate of uptake was substantially attenuated, with only 2.47 +/- 0.2 (mean +/- S.D, n = 3) nmol Ca2+/mg protein sequestered in 60 min. Ca2+ sequestration was 93% inhibited by 180 mM NaCl [I50% of 78.7 +/- 9.3 mM NaCl (mean +/- S.D., n = 11)] but only slightly inhibited by KCl or MgCl2. Ca2+ sequestration was not stimulated by incubation with MgATP but was inhibited by 57% after incubation with 30 microM monensin. Ca2+ sequestration was dependent on extravesicular Ca2+ with half-maximal sequestration at pCa2+ 6.81 +/- 0.028 (mean +/- S.D., n = 3). Sequestered Ca2+ could be exchanged with external 45Ca2+, the exchange rate was first order (k of 0.042 +/- 0.004: mean +/- S.D., n = 3) and saturated at 27.7 +/- 1.1 nmol Ca2+/mg (mean +/- S.D., n = 3). The Ca2+/Ca2+ exchange system was totally inhibited by NaCl or KCl but only slightly by MgCl2. About 75% of sequestered 45Ca2+ could be released by incubation with NaCl, but only 8% was released by incubation with KCl. Half-maximal release of sequestered 45Ca2+ required 69.3 +/- 12.2 mM NaCl (mean +/- S.D., n = 3). The Na+-induced release of sequestered 45Ca2+ was rapid, t0.5 of 2.80 +/- 0.63 min (mean +/- S.D., n = 3) and inhibited at 4 degrees C. The concurrent incubation of chromaffin granules with 45Ca2+ and either annexin proteins V or VI resulted in attenuated uptake of 45Ca2+. These results suggest that Ca2+ uptake in adrenal chromaffin granules is regulated by Na+ and Ca2+ gradients and also possibly by annexins V and VI.
通过等密度的甲泛影酰胺梯度离心法纯化牛嗜铬细胞分泌颗粒，并对其Ca2+ 螯合途径进行了表征。37℃时Ca2+ 螯合速率呈一级反应，最大摄取量为26.9±0.46（平均值±标准差，n = 3）nmol Ca2+/mg蛋白质，一级速率常数（k）为0.046±0.002 min-1。4℃时摄取速率显著降低，60分钟内仅螯合2.47±0.2（平均值±标准差，n = 3）nmol Ca2+/mg蛋白质。180 mM NaCl可抑制93%的Ca2+ 螯合 [半数抑制浓度（I50%）为78.7±9.3 mM NaCl（平均值±标准差，n = 11）]，但KCl或MgCl2仅产生轻微抑制。与MgATP孵育不会刺激Ca2+ 螯合，但与30 μM莫能菌素孵育后会抑制57%。Ca2+ 螯合依赖于囊泡外Ca2+，在pCa2+ 6.81±0.028（平均值±标准差，n = 3）时达到半数最大螯合。螯合的Ca2+ 可与外部45Ca2+ 交换，交换速率为一级反应（k为0.042±0.004：平均值±标准差，n = 3），在27.7±1.1 nmol Ca2+/mg（平均值±标准差，n = 3）时达到饱和。Ca2+/Ca2+ 交换系统完全被NaCl或KCl抑制，但仅被MgCl2轻微抑制。约75%螯合的4,5Ca2+ 可通过与NaCl孵育释放，但与KCl孵育仅释放8%。螯合的45Ca2+ 半数最大释放需要69.3±12.2 mM NaCl（平均值±标准差，n = 3）。Na+ 诱导的螯合45Ca2+ 释放迅速，半衰期（t0.5）为2.80±0.63分钟（平均值±标准差，n = 3），并在4℃时受到抑制。嗜铬颗粒与45Ca2+ 以及膜联蛋白V或VI同时孵育会导致45Ca2+ 摄取减少。这些结果表明，肾上腺嗜铬颗粒中的Ca2+ 摄取受Na+ 和Ca2+ 梯度调节，也可能受膜联蛋白V和VI调节。
