Shafir I, Feng W, Shoshan-Barmataz V
Department of Life Sciences and the Zlotowski Center for Neuroscience, Ben Gurion University of the Negev, Beer Sheva, Israel.
J Bioenerg Biomembr. 1998 Oct;30(5):499-510. doi: 10.1023/a:1020598315287.
In this study, we purified and characterized the voltage-dependent anion channel (VDAC) from the Torpedo electric organ. Using immunogold labeling, VDAC was colocalized with the voltage-gated Ca2+ channel in the synaptic plasma membrane. By immunoblot analysis, five protein bands in synaptosomes isolated from the Torpedo electric organ cross reacted with two monoclonal anti-VDAC antibody. No more than about 7 to 10% mitochondrial contains could be detected in any synaptosomal membrane preparation tested. This was estimated by comparing the specific activity in mitochondria and synaptosomes of succinate-cytochrome-c oxidoreductase and antimycin-insensitive NADH-cytochrome-c oxidoreductase activities; mitochondrial inner and outer membrane marker enzymes, respectively. [14C]DCCD (dicyclohexylcarbodiimide), which specifically label mitochondrial VDAC, labeled four 30-35 kDa protein bands that were found to interact with the anti-VDAC antibody. The distribution of the Torpedo VDAC protein bands was different among membranes isolated from various tissues. VDAC was purified from synaptosomes and a separation between two of the proteins was obtained. The two purified proteins were characterized by their single channel activity and partial amino acid sequences. Upon reconstitution into a planar lipid bilayer, the purified VDACs showed voltage-dependent channel activity with properties similar to those of purified mitochondrial VDAC. Amino acid sequence of four peptides, derived from VDAC band II, exhibited high homology to sequences present in human VDACI (98%), VDAC2 (91.8%), and VDAC3 (90%), while another peptide, derived from VDAC band III, showed lower homology to either VDAC1 (88.4%) or VDAC2 (79%). Two more peptides show high homology to the sequence present in mouse brain VDAC3 (100 and 78%). In addition, we demonstrate the translocation of ATP into synaptosomes, which is inhibited by DCCD and by the anion transport inhibitor DIDS. The possible function of VDAC in the synaptic plasma membrane is discussed.
在本研究中，我们从电鳐电器官中纯化并鉴定了电压依赖性阴离子通道（VDAC）。使用免疫金标记法，VDAC与突触质膜中的电压门控Ca2+通道共定位。通过免疫印迹分析，从电鳐电器官分离的突触体中的五条蛋白带与两种抗VDAC单克隆抗体发生交叉反应。在任何测试的突触体膜制剂中，检测到的线粒体含量不超过约7%至10%。这是通过比较琥珀酸 - 细胞色素c氧化还原酶和抗霉素不敏感的NADH - 细胞色素c氧化还原酶（分别为线粒体内膜和外膜标记酶）在线粒体和突触体中的比活性来估计的。特异性标记线粒体VDAC的[14C]DCCD（二环己基碳二亚胺）标记了四条30 - 35 kDa的蛋白带，这些蛋白带被发现与抗VDAC抗体相互作用。电鳐VDAC蛋白带在从不同组织分离的膜中的分布有所不同。从突触体中纯化出VDAC，并实现了两种蛋白之间的分离。通过单通道活性和部分氨基酸序列对这两种纯化蛋白进行了鉴定。重新组装到平面脂质双分子层后，纯化的VDAC表现出电压依赖性通道活性，其特性与纯化的线粒体VDAC相似。源自VDAC带II 的四个肽段的氨基酸序列与人类VDAC1（98%）、VDAC2（91.8%）和VDAC3（90%）中的序列具有高度同源性，而源自VDAC带III 的另一个肽段与VDAC1（88.4%）或VDAC2（79%）的同源性较低。另外两个肽段与小鼠脑VDAC3中的序列具有高度同源性（分别为100%和78%）。此外，我们证明了ATP向突触体的转运受到DCCD和阴离子转运抑制剂DIDS的抑制。文中讨论了VDAC在突触质膜中的可能功能。
