Jentsch T J, Garcia A M, Lodish H F
Whitehead Institute for Biomedical Research, 9 Cambridge Center, MA 02142.
Biochem J. 1989 Jul 1;261(1):155-66. doi: 10.1042/bj2610155.
Polyclonal rabbit antibodies were raised against 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS), an inhibitor of a variety of anion transport proteins. These antibodies specifically recognize SITS-reacted erythrocyte band 3 in immunoprecipitations and Western blots. In Western blots of SITS-reacted membrane proteins derived from vesicles of the electric organ of Torpedo californica (known to express a SITS-sensitive Cl- channel) the antibodies recognized two major species of approximately 93 kDa and approximately 105 kDa. The approximately 93 kDa protein was identified as the alpha-subunit of the Na,K-ATPase. The approximately 105 kDa protein (designated sp105) is a glycoprotein which binds to wheat-germ agglutinin and concanavalin A and is present as a disulphide-linked homodimer under non-reducing conditions. A partial amino acid sequence and a polyclonal antibody were used to clone the corresponding cDNA. sp105 is encoded in electroplax by two abundant mRNAs of approximately 6 and approximately 6.8 kb. A hybridizing mRNA of approximately 5 kb was over 200-fold and over 500-fold less abundant in brain and heart respectively. Sequence analysis of the cDNA predicted a novel protein of 697 amino acids containing eight potential N-linked glycosylation sites. Analysis of hydrophobicity indicated the presence of at least one, and possibly three, putative membrane-spanning domains. When expressed from the Sp6 message in Xenopus laevis oocytes, the protein was inserted into membranes, glycosylated and processed to form a dimer. However, no increase in 36Cl uptake or in membrane conductance could be detected. We found no effect of hybrid depleting the specific message on expression of the Torpedo electroplax Cl- channel in oocytes. Thus we conclude that this novel electroplax membrane protein is probably not a functional part of the chloride channel.
用4-乙酰氨基-4'-异硫氰基芪-2,2'-二磺酸（SITS，多种阴离子转运蛋白的抑制剂）免疫家兔制备多克隆抗体。这些抗体在免疫沉淀和蛋白质印迹中能特异性识别与SITS反应的红细胞带3。在对来自加州电鳐电器官囊泡（已知表达对SITS敏感的氯离子通道）的与SITS反应的膜蛋白进行蛋白质印迹时，抗体识别出两种主要条带，分子量分别约为93 kDa和约105 kDa。约93 kDa的蛋白被鉴定为钠钾ATP酶的α亚基。约105 kDa的蛋白（命名为sp105）是一种糖蛋白，可与麦胚凝集素和伴刀豆球蛋白A结合，在非还原条件下以二硫键连接的同型二聚体形式存在。利用部分氨基酸序列和多克隆抗体克隆了相应的cDNA。sp105在电板中由两种丰度较高的mRNA编码，大小分别约为6 kb和约6.8 kb。一条约5 kb的杂交mRNA在脑和心脏中的丰度分别比电板中低200倍和500倍以上。对cDNA的序列分析预测了一种由697个氨基酸组成的新蛋白，含有8个潜在的N-连接糖基化位点。疏水性分析表明存在至少一个，可能是三个推定的跨膜结构域。当在非洲爪蟾卵母细胞中从Sp6信使RNA表达时，该蛋白插入膜中，进行糖基化并加工形成二聚体。然而，未检测到36Cl摄取或膜电导增加。我们发现杂交耗尽特异性信使RNA对卵母细胞中电鳐电板氯离子通道的表达没有影响。因此我们得出结论，这种新的电板膜蛋白可能不是氯离子通道的功能组成部分。
