THE INTERNAL PH AND MEMBRANE-POTENTIAL OF THE INSULIN-SECRETORY GRANULE
THE INTERNAL PH AND MEMBRANE-POTENTIAL OF THE INSULIN-SECRETORY GRANULE
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DOI：
10.1042/bj2040171
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发表时间：
1982-01-01
影响因子：
4.1
通讯作者：
HUTTON, JC
中科院分区：
文献类型：
--
作者：
HUTTON, JC
通过对渗透阴离子硫醇[14C]氰酸酯或渗透碱[14C]甲胺的摄取，在体外测定了大鼠胰岛素分泌颗粒膜电位（. delta .PSI.）和pH梯度（. delta .pH .）。新鲜制备的颗粒在含有蔗糖和低浓度缓冲盐的等渗透培养基中培养，表现出酸性的内部pH值和。delta…PSI。积极的内部。在这些条件下添加MgATP2-没有改变。delta。pH值，但是产生了显著的δ…PSI…相反，当渗透阴离子也包括在内，ATP产生显著增加。delta。pH和较小的增量。delta…PSI…NH4+盐还原了。delta。颗粒膜上的pH值。在ATP存在的情况下，这种效应伴随着delta…PSI…的倒数增加。当尼日利亚菌素与K+一起添加或降低介质pH时，类似的互惠性很明显，表明这些梯度通过共同的电生成过程联系在一起。质子载体羰基氰化物对三氟甲氧基苯腙、万霉素、尼日尔霉素和K+的组合以及依赖于Mg2+的ATP酶抑制剂三丁基锡可以逆转ATP的作用。低温破坏颗粒膜或低渗透孵育条件也显著减少了14c标记示踪分子的摄取。这些结果很容易在化学渗透假说中解释，该假说提出胰岛素颗粒具有向内定向的电致质子易位Mg2+依赖性atp酶，并附加假设膜具有低质子渗透率。体内颗粒内pH值估计在5到6之间。该值对应于锌-胰岛素六聚体结晶的最佳条件。然而，可以设想与胰岛素颗粒内化学渗透过程有关的其他几个功能。
The membrane potential (.DELTA..PSI.) and the pH gradient (.DELTA.pH) across the membrane of the rat insulin-secretory granule were determined in studies in vitro from the uptake of the permeant anion thiol[14C]cyanate or the permeant base [14C]methylamine. Freshly prepared granules incubated in iso-osmotic medium containing sucrose and low concentrations of buffer salts exhibited an acidic internal pH and a .DELTA..PSI. positive inside. Addition of MgATP2- under these conditions did not alter the .DELTA.pH, but produced a marked increase in the .DELTA..PSI.. Conversely, when a permeant anion was also included, ATP produced a marked increase in the .DELTA.pH and a lesser increment in the .DELTA..PSI.. NH4+ salts reduced the .DELTA.pH across granule membranes. In the presence of ATP this effect was accompanied by a reciprocal increase in the .DELTA..PSI.. A similar reciprocity was evident when nigericin was added together with K+ or on decreasing the medium pH, suggesting that these gradients were linked by a common electrogenic process. The effects of ATP were reversed by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, the combination of valinomycin, nigericin and K+, and by the Mg2+-dependent ATPase inhibitor tributyltin. Uptakes of 14C-labeled tracer molecules were also markedly reduced by cryogenic disruption of the granule membrane or hypo-osmotic incubation conditions. These results were readily interpreted within a chemiosmotic hypothesis, which proposed that the insulin granules possess an inwardly-directed electrogenic proton-translocating Mg2+-dependent ATPase with the additional postulate that the membrane has a low proton permeability. The intragranular pH was estimated as being between 5 and 6 in vivo. Such a value corresponds to optimal conditions for the crystallization of Zn-insulin hexamers. Several other functions related to chemiosmotic processes within insulin granules, however, may be envisaged.