Cleves A E, Clift-O'Grady L, Kelly R B
Department of Biochemistry & Biophysics, University of California, San Francisco 94143-0534, USA.
Neurochem Res. 1997 Aug;22(8):933-40. doi: 10.1023/a:1022462523315.
Synaptic vesicles are released from membranes during incubation at 37 degrees C in the presence of ATP (adenosine triphosphate). The donor membranes are a rapidly sedimenting fraction derived from the neuroendocrine cell line PC12 (pheochromocytoma 12). These starting membranes contain the synaptic vesicle proteins, synaptophysin and SV2, and the endosomal markers transferrin receptor and cation-independent MPR (mannose 6-phosphate receptor). Incubating the membranes in vitro increased the amount of organelles that migrate as synaptic vesicles in velocity sedimentation gradients. The synaptic vesicle fractions that contain both synaptophysin and SV2 do not contain endosomal markers. A synaptic vesicle increase in vitro is time-, cytosol-, ATP- and temperature-dependent and is inhibited by NEM (N-ethylmaleimide), BFA (brefeldin A) and aluminum fluoride, but not GTP gamma S (guanosine-5'O-C3-thiotriphosphate). The production of synaptic vesicles under these conditions is unlike the de novo generation of vesicles from endosomes (1). Incubation in vitro under the conditions described here may allow the final stages of synaptic vesicle formation, uncoating or undocking, to occur but not the initiation of formation de novo.
在37摄氏度、存在三磷酸腺苷（ATP）的条件下孵育时，突触小泡从膜上释放出来。供体膜是源自神经内分泌细胞系PC12（嗜铬细胞瘤12）的一个快速沉降组分。这些起始膜含有突触小泡蛋白、突触素和SV2，以及内体标记物转铁蛋白受体和不依赖阳离子的甘露糖6-磷酸受体（MPR）。在体外孵育这些膜会增加在速度沉降梯度中作为突触小泡迁移的细胞器数量。同时含有突触素和SV2的突触小泡组分不含有内体标记物。体外突触小泡的增加是时间、胞质溶胶、ATP和温度依赖性的，并且受到N-乙基马来酰亚胺（NEM）、布雷菲德菌素A（BFA）和氟化铝的抑制，但不受5'-O-（3-硫代三磷酸）鸟苷（GTPγS）的抑制。在这些条件下突触小泡的产生不同于从内体从头生成小泡（1）。在此处所述条件下进行体外孵育可能会使突触小泡形成、脱包被或脱离的最后阶段发生，但不会发生从头形成的起始阶段。
