IDENTIFICATION OF THE ANION-EXCHANGE PROTEIN OF EHRLICH CELLS - A KINETIC-ANALYSIS OF THE INHIBITORY EFFECTS OF 4,4'-DIISOTHIOCYANO-2,2'-STILBENE-DISULFONIC ACID (DIDS) AND LABELING OF MEMBRANE-PROTEINS WITH H-3 DIDS
IDENTIFICATION OF THE ANION-EXCHANGE PROTEIN OF EHRLICH CELLS - A KINETIC-ANALYSIS OF THE INHIBITORY EFFECTS OF 4,4'-DIISOTHIOCYANO-2,2'-STILBENE-DISULFONIC ACID (DIDS) AND LABELING OF MEMBRANE-PROTEINS WITH H-3 DIDS
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DOI：
10.1007/bf01869388
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发表时间：
1986-01-01
影响因子：
2.4
通讯作者：
HOFFMANN, EK
中科院分区：
文献类型：
--
作者：
JESSEN, F;SJOHOLM, C;HOFFMANN, EK
在艾利希腹水肿瘤细胞中，4,4''-二异硫氰基-2,2''-二苯乙烯二磺酸 (DIDS) 可逆和不可逆地抑制氯离子交换。可逆抑制实际上是瞬时的并且具有竞争性质，在零氯化物浓度下Ki约为2μM。这通过 DIDS 与转运蛋白的缓慢不可逆结合而成功，氯依赖性表明与可逆 DIDS 结合/抑制相同的位点结合。为了鉴定参与阴离子交换的膜蛋白，用 3H-DIDS 标记细胞。当氯化物浓度较低(15 mM)时，将细胞与25 μM DIDS在pH 8.2下孵育10分钟导致对DIDS敏感的氯化物交换通量的抑制超过95%。该条件用于 3H-DIDS 标记实验。温育后，破碎细胞，分离并溶解膜，并通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳分离蛋白质。凝胶中 3H 活性的分布仅显示一个主峰，这可能与分子量约为 30,000 道尔顿的蛋白质有关。每个细胞的传输位点数量估计约为 400,000 个，根据稳态条件下的 DIDS 敏感氯通量，我们计算出每个位点每秒 340 个离子的周转数。
In Ehrlich ascites tumor cells 4,4''-diisothiocyano-2,2''-stilbene-disulfonic acid (DIDS) inhibits the chloride exchange both reversibly and irreversibly. The reversible inhibition is practically instantaneous and of a competitive nature with Ki about 2 .mu.M at zero chloride concentration. This is succeeded by a slow irreversible binding of DIDS to the transporter, with a chloride dependence suggesting binding to the same site as for reversible DIDS binding/inhibition. To identify the membrane protein involved in anion exchange, cells were labeled with 3H-DIDS. Incubation of cells for 10 min with 25 .mu.M DIDS at pH 8.2 leads to more than 95% inhibition of the DIDS-sensitive chloride exchange flux when the chloride concentration is low (15 mM). This condition was used for the 3H-DIDS-labeling experiments. After incubation the cells were disrupted, the membranes isolated and solubilized, and the proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The distribution of the 3H-activity in the gel showed only one major peak, which could be related to protein with a mol wt of about 30,000 Daltons. The number of transport sites was estimated at about 400,000 per cell, and from the DIDS-sensitive chloride flux under steady-state conditions we calculate a turnover number of 340 ions per sec per site.