Soluble N-ethylmaleimide-sensitive-factor attachment protein and N-ethylmaleimide-insensitive factors are required for Ca2+-stimulated exocytosis of insulin.
Soluble N-ethylmaleimide-sensitive-factor attachment protein and N-ethylmaleimide-insensitive factors are required for Ca2+-stimulated exocytosis of insulin.
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Ca2 刺激胰岛素胞吐作用需要可溶性 N-乙基马来酰亚胺敏感因子附着蛋白和 N-乙基马来酰亚胺不敏感因子。
作者：
C. E. Kiraly;A. Morgan;R. Burgoyne;U. Weller;C. Wollheim;J. Lang
Ca2+ 刺激透化胰岛素分泌细胞的胞吐作用。为了研究参与 Ca2+ 反应的假定胞质成分，使用链球菌溶血素-O 对 HIT-T15 细胞（一种胰腺 B 细胞系）进行透化，该过程允许快速交换包括大分子在内的可溶性成分。我们发现，在该细胞制剂中，对 Ca2+ 的分泌反应，但对 5'-γ-硫代]三磷酸鸟苷的分泌反应，随着时间的推移而消失，并且可以通过大鼠脑细胞质以浓度依赖性方式恢复。在高分子量热不稳定部分 N-乙基马来酰亚胺 (NEM) 敏感组分中发现了大鼠脑细胞质的重构活性。发现 NEM 敏感因子 (NSF) 和可溶性 NSF 附着蛋白 (α-SNAP) 在 HIT-T15 细胞中表达，并在穿孔细胞中大量丢失（剩余约 30%）。当添加到透化细胞中时，重组 α-SNAP 部分重建了 Ca2+ 反应。此外，α-SNAP 将 NEM 处理的细胞质的效果恢复到未处理的细胞质中观察到的水平。相反，NSF 在单独预孵育或与 NEM 处理的胞质溶胶一起预孵育时无效。我们的结果表明，α-SNAP 和 NEM 不敏感的胞质因子均参与内分泌 HIT-T15 细胞的 Ca2+ 介导的胞吐作用。
Ca2+ stimulates exocytosis in permeabilized insulin-secreting cells. To investigate the putative cytosolic components involved in the Ca2+ response, HIT-T15 cells (a pancreatic B-cell line) were permeabilized with streptolysin-O, a procedure that allows rapid exchange of soluble components including macromolecules. We found that in this cell preparation the secretory response to Ca2+ but not to guanosine 5'-[gamma-thio]triphosphate was lost as a function of time and could be restored by rat brain cytosol in a concentration-dependent manner. Reconstitutive activity of rat brain cytosol was found in a high-molecular-mass heat-labile partially N-ethylmaleimide(NEM)-sensitive fraction. The NEM-sensitive factor (NSF) and the soluble NSF attachment protein (alpha-SNAP) were found to be expressed in HIT-T15 cells and largely lost (about 30% remaining) from porated cells. Recombinant alpha-SNAP partially reconstituted the Ca2+ response when added to the permeabilized cells. Moreover, alpha-SNAP restored the effect of NEM-treated cytosol to the level observed for untreated cytosol. In contrast, NSF was ineffective when preincubated alone or with NEM-treated cytosol. Our results indicate that both alpha-SNAP and NEM-insensitive cytosolic factors are involved in Ca2+-mediated exocytosis from endocrine HIT-T15 cells.