Voltage-Dependent Anion Channel Proteins in Synaptosomes of the Torpedo Electric Organ: Immunolocalization, Purification, and Characterization
Voltage-Dependent Anion Channel Proteins in Synaptosomes of the Torpedo Electric Organ: Immunolocalization, Purification, and Characterization
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DOI：
10.1023/a:1020598315287
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发表时间：
1998-10
影响因子：
3
通讯作者：
I. Shafir;W. Feng;Varda Shoshan-Barmataz
中科院分区：
文献类型：
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作者：
I. Shafir;W. Feng;Varda Shoshan-Barmataz
在这项研究中，我们从鱼雷的电器官中纯化并表征了电压依赖性阴离子通道（VDAC）。利用免疫金标记，VDAC与突触质膜上电压门控的Ca2+通道共定位。通过免疫印迹分析，从鱼雷电器官中分离的突触小体的5个蛋白带与2个单克隆抗vdac抗体发生交叉反应。在所测试的任何突触体膜制剂中检测到的线粒体含量不超过约7 - 10%。这是通过比较线粒体和突触体中琥珀酸-细胞色素-氧化还原酶和抗霉素不敏感的nadh -细胞色素-氧化还原酶的活性来估计的；线粒体内外膜标记酶。[14C]专门标记线粒体VDAC的DCCD （dicyclohexylcarbodiimide）标记了与抗VDAC抗体相互作用的4个30-35 kDa蛋白带。鱼雷VDAC蛋白带在不同组织分离膜上的分布是不同的。从突触体中纯化了VDAC，并获得了两种蛋白之间的分离。这两个纯化蛋白具有单通道活性和部分氨基酸序列。在重组成平面脂质双分子层后，纯化的VDAC显示出与纯化的线粒体VDAC相似的电压依赖性通道活性。来自VDAC II带的4个肽的氨基酸序列与人类VDAC1（98%）、VDAC2（91.8%）和VDAC3（90%）的序列具有较高的同源性，而来自VDAC III带的另一个肽的氨基酸序列与VDAC1（88.4%）或VDAC2（79%）的同源性较低。另外两个多肽与小鼠脑VDAC3中存在的序列高度同源（分别为100%和78%）。此外，我们还证明了ATP在突触体中的易位被DCCD和阴离子运输抑制剂DIDS所抑制。讨论了VDAC在突触质膜中的可能作用。
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-coxidoreductase and antimycin-insensitive NADH–cytochrome-coxidoreductase 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 VDAC1 (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.