Molecular and functional characterization of VDAC2 purified from mammal spermatozoa
Molecular and functional characterization of VDAC2 purified from mammal spermatozoa
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DOI：
10.1042/bsr20080123
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发表时间：
2009-12-01
影响因子：
4
通讯作者：
Hinsch, Elvira
中科院分区：
文献类型：
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作者：
Menzel, Viviana A.;Cassara, M. Carolina;Hinsch, Elvira
电压依赖性阴离子通道(VDAC)是一种位于线粒体膜外膜的致孔蛋白。在高等真核生物中，有三个基因编码VDAC。然而，对VDAC亚型的了解主要局限于VDAC1，这是迄今为止从活组织中鉴定的唯一一种亚型。我们以牛精子为原料，对VDAC2亚型进行了高度浓缩。VDAC2是在Triton X-100增溶的精子蛋白的羟基磷灰石/硅藻石穿透过程中得到的。该组分在相对分子质量为30-35 kDa的范围内表现为两条主带和一条微弱带。双向电泳以不同的考马斯亮蓝染色强度在10个点上分辨出这些条带。每一种亚型的单特异性抗体的Western-印迹分析和MS多肽序列分析表明，在电泳法中主要分解的蛋白是VDAC2，其他亚型也有少量的污染。对高相对分子质量的VDAC2蛋白的蛋白质组学分析表明，在同一材料中，除A(24)AR(26)三肽外，还存在两个可能的氨基酸替换(T-88到L-88和A(97)到Q(97))。平面脂质双层中VDAC2孔的重建显示出典型的线粒体孔蛋白特征。观察到膜电导的逐步增加，主要电导约为。3.5毫微秒(纳米西门子)在1MKCl.通常，可以观察到小而短暂的波动，单通道电导约为。1.5纳秒。牛精子VDAC2对阴离子具有选择性，表现出电压依赖性。本研究是首次报道从哺乳动物组织中纯化和鉴定VDAC2的工作。
VDAC (voltage-de pendent anion channel) is the pore-forming protein located in the outer mitochondrial membrane. In higher eukaryotes, three genes encode VDAC. Nevertheless, the knowledge of VDAC isoforms is mainly restricted to VDAC1, the only isoform that has been characterized from living tissues to date. We have highly enriched the isoform VDAC2 using as starting material bovine spermatozoa. VDAC2 was obtained in the hydroxyapatite/celite pass-through of sperm proteins solubilized with Triton X-100. This fraction showed in SDS/PAGE two major bands and one faint band in the molecular mass range of 30-35 kDa. Two-dimensional electrophoresis resolved these bands in ten spots with various Coomassie Blue staining intensities. Western-blot analysis with antibodies monospecific for each isoform and MS peptide sequencing showed that the main protein resolved in electrophoresis was VDAC2 with minor contaminations of the other isoforms. Proteomic analysis of the higher molecular mass VDAC2 protein allowed the coverage of the whole protein with the exception of the tripeptide A(24)AR(26), In the same material, the presence of two possible amino acid substitutions (T-88 to L-88 and A(97) to Q(97)) was revealed. Reconstitution of VDAC2 pores in planar lipid bilayers showed typical features of mitochondrial porins. Stepwise increases in membrane conductance were observed with a predominant conductance of approx. 3.5 nS (nanoSiemens) in 1 M KCl. Very often, small short-lived fluctuations were observed with single-channel conductance of approx. 1.5 nS. Bovine spermatozoa VDAC2 was anion selective and showed voltage dependence. The present study is the first work to report the purification and characterization of VDAC2 from a mammalian tissue.