Protein phosphorylation in permeabilized pancreatic islet cells.
Protein phosphorylation in permeabilized pancreatic islet cells.
复制标题
透化胰岛细胞中的蛋白质磷酸化。
作者：
J. Colca;B. Wolf;P. Comens;M. Mcdaniel
毛地黄皂苷透化的胰岛细胞系统的开发，以表征Ca 2+和钙调蛋白依赖性蛋白磷酸化进一步，并确定是否激活这种膜结合的过程是足够的启动Ca 2+刺激的胰岛素分泌。通过台盼蓝排除法、乳酸脱氢酶的细胞外渗漏和对[γ-32 P]ATP的渗透性来评估毛地黄皂苷透化质膜的功效。这种处理没有可检测地改变透化细胞的超微结构。毛地黄皂苷是同样有效的，当提交给胰岛细胞，以前已经分散或直接完整的分离胰岛。Ca 2+和钙调素依赖性磷酸化的内源性膜结合底物可以证明在透化细胞与[γ-32 P]ATP孵育。这种活性显示的特征是类似于那些描述的蛋白激酶在亚细胞组分中测量，并依赖于添加外源性钙调蛋白，表明钙调蛋白已被删除的激酶通过透化的细胞。证明了毛地黄皂苷透化胰岛的钙依赖性胰岛素释放，在0.1 μ M游离Ca 2+时发生半数最大释放，在0.2 μ M游离Ca 2+时发生最大分泌。在这些条件下，钙调素没有进一步增强胰岛素的释放，虽然钙调素的刺激作用，观察到游离Ca 2+的情况下。这些研究表明，透化胰岛模型将是有用的解剖出参与钙激活的胰岛素分泌的因素。
A system of digitonin-permeabilized islet cells was developed to characterize Ca2+- and calmodulin-dependent protein phosphorylation further and to determine whether activation of this membrane-bound process was sufficient for initiation of Ca2+-stimulated insulin secretion. The efficacy of digitonin in permeabilizing the plasma membrane was assessed by Trypan Blue exclusion, by extracellular leakage of lactate dehydrogenase, and by permeability to [gamma-32P]ATP. This treatment did not detectably alter the ultrastructure of the permeabilized cells. Digitonin was equally effective when presented to islet cells that had been previously dispersed or directly to intact isolated islets. The Ca2+- and calmodulin-dependent phosphorylation of endogenous membrane-bound substrates could be demonstrated in the permeabilized cells incubated with [gamma-32P]ATP. This activity displayed characteristics that were similar to those described for the protein kinase measured in subcellular fractions and was dependent on addition of exogenous calmodulin, indicating that calmodulin had been removed from the kinase by permeabilization of the cells. Ca2+-dependent insulin release by the digitonin-permeabilized islet was demonstrated, with half-maximal release occurring at 0.1 microM-free Ca2+ and maximal secretion at 0.2 microM-free Ca2+. Under these conditions, calmodulin did not further enhance insulin release, although a stimulatory effect of calmodulin was observed in the absence of free Ca2+. These studies indicate that the permeabilized-islet model will be useful in dissecting out the factors involved in Ca2+-activated insulin secretion.