Trikash I O, Volynets G P, Remenyak O V, Gorchev V F
National Academy of Sciences of Ukraine, Palladin Institute of Biochemistry, Department of Neurochemistry, Leontovich st. 9, 01601 Kiev, Ukraine.
Neurochem Int. 2008 Dec;53(6-8):401-7. doi: 10.1016/j.neuint.2008.09.010. Epub 2008 Sep 24.
The present study involves the testing and characterization of synaptic vesicle (SV) docking and fusion as the steps of exocytosis using two different approaches in vitro. The interaction of SVs was determined by the changing of particles size in suspensions by the method of dynamic light scattering (DLS). Fluorescence assay is represented for studying the mechanism of SV membrane fusion. The sizes of membrane particles were shown to increase in the medium containing cytoplasmic proteins of synaptosomes. Therefore, the cytosolic proteins are suggested to promote the SVs into close proximity where they may become stably bound or docked. The specific effect of synaptosomal cytosolic proteins on the interaction of SVs in the cell-free system was demonstrated. The incubation of SVs with liver cytosol proteins or in the bovine serum albumin solution did not lead to the enlargement of the particles size. The fusion reaction of the SVs membranes occurred within the micromolar range of Ca(2+) concentrations. Our studies have shown that in vitro process of exocytosis can be divided into Ca(2+)-independent step, termed docking and followed by fusion step that is triggered by Ca(2+). The role of cytosolic proteins of synaptosomes in docking and fusion of SVs in cell-free system was further confirmed.
本研究涉及利用两种不同的体外方法，对作为胞吐作用步骤的突触小泡（SV）对接和融合进行测试与表征。通过动态光散射（DLS）方法，根据悬浮液中颗粒大小的变化来确定SV之间的相互作用。采用荧光测定法来研究SV膜融合的机制。结果显示，在含有突触体细胞质蛋白的培养基中，膜颗粒的大小会增加。因此，推测胞质蛋白可促使SV紧密靠近，使其可能稳定结合或对接。证实了突触体胞质蛋白在无细胞系统中对SV相互作用的特定影响。将SV与肝细胞溶质蛋白一起孵育或置于牛血清白蛋白溶液中，并不会导致颗粒大小增大。SV膜的融合反应发生在微摩尔浓度范围的Ca(2+) 内。我们的研究表明，体外胞吐过程可分为不依赖Ca(2+) 的步骤，即对接，随后是由Ca(2+) 触发的融合步骤。进一步证实了突触体胞质蛋白在无细胞系统中对SV对接和融合的作用。
