Zheng X, Bobich J A
Department of Chemistry, Texas Christian University, Fort Worth 76129, USA.
Biochemistry. 1998 Sep 8;37(36):12569-75. doi: 10.1021/bi980651w.
In streptolysin-O (SLO)-perforated rat brain cortical synaptosomes, Ca2+-induced [3H]noradrenaline (3H-NA) release began with a phase lasting about 1 min that did not depend on MgATP. Subsequent release became increasingly MgATP-dependent. The first phase involved release from previously "primed" synaptic vesicles. MgATP-dependent release, on the other hand, was release from unprimed vesicles that needed to be primed by ATP hydrolysis before they could be fused with the presynaptic membrane. Vesicle depriming was detected by observing that the initial release decreased when the synaptosomes were perforated and incubated for 2 min in the absence of MgATP before increasing Ca2+ to promote release. One millimolar N-ethylmaleimide (NEM) inhibited both MgATP-dependent and MgATP-independent release at all times of incubation (0.5-5 min), and inhibition by NEM was partially reversed at short (0.5 min) and longer (5 min) times by adding intact N-ethylmaleimide sensitive fusion protein (NSF) to the perforated synaptosomes. Polyclonal antibodies against the N-terminal domain of NSF produced dose-dependent inhibition of Ca2+-induced 3H-NA release. This inhibition occurred in both early and late release phases and was highly significant at early times if the perforated synaptosomes were preincubated for 2 min with anti-NSF. These results indicate participation of NSF both after vesicular fusion, probably for separation of SNARE proteins in v/t-SNARE complexes before endocytosis, and, surprisingly, after docking, possibly to maintain vesicles in a primed state and reverse depriming during regulated secretion.
在经链球菌溶血素-O（SLO）穿孔处理的大鼠脑皮质突触体中，Ca2+诱导的[3H]去甲肾上腺素（3H-NA）释放开始时存在一个持续约1分钟的阶段，该阶段不依赖于MgATP。随后的释放则越来越依赖于MgATP。第一阶段涉及从先前“致敏”的突触小泡中释放。另一方面，依赖MgATP的释放是从未致敏的小泡中释放，这些小泡需要通过ATP水解进行致敏，才能与突触前膜融合。通过观察到当突触体被穿孔并在无MgATP的情况下孵育2分钟，然后增加Ca2+以促进释放时，初始释放减少，从而检测到小泡的去致敏。1毫摩尔N-乙基马来酰亚胺（NEM）在孵育的所有时间（0.5 - 5分钟）均抑制依赖MgATP和不依赖MgATP的释放，并且在短时间（0.5分钟）和长时间（5分钟）时，通过向穿孔的突触体中添加完整的N-乙基马来酰亚胺敏感融合蛋白（NSF），NEM的抑制作用可部分逆转。针对NSF N端结构域的多克隆抗体对Ca2+诱导的3H-NA释放产生剂量依赖性抑制。这种抑制在释放的早期和晚期阶段均发生，如果穿孔的突触体与抗NSF预孵育2分钟，则在早期阶段具有高度显著性。这些结果表明，NSF不仅在小泡融合后参与其中，可能用于在内吞作用之前分离v/t-SNARE复合物中的SNARE蛋白，而且令人惊讶的是，在对接后也参与其中，可能是为了在调节性分泌过程中将小泡维持在致敏状态并逆转去致敏。
