Chemical modification and labeling of glutamate residues at the stilbenedisulfonate site of human red blood cell band 3 protein.
Chemical modification and labeling of glutamate residues at the stilbenedisulfonate site of human red blood cell band 3 protein.
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DOI：
10.1016/s0021-9258(19)75693-2
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发表时间：
1987-02
期刊：
影响因子：
--
通讯作者：
M. Jennings;M. Anderson
中科院分区：
文献类型：
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作者：
M. Jennings;M. Anderson
发展了一种新的化学修饰和标记蛋白质羧基的方法。羧基用伍德沃德试剂K（N-乙基-5-苯基异恶唑鎓 3 '-磺酸盐）活化，加合物用[3 H] BH 4还原。该方法已被应用于人红细胞的阴离子转运蛋白（带3）。Woodward试剂K是条带3介导的阴离子转运的合理有效的抑制剂;在pH 6.5下将完整细胞暴露于2 mM试剂5分钟产生80%的转运抑制。这种抑制作用是可被4，4 '-二硝基芪-2，2'-二磺酸盐保护的残基修饰的结果。用伍德沃德试剂K处理完整细胞，然后用B3 H4处理，导致条带3的广泛标记，而细胞内蛋白质如血影蛋白的标记最少。蛋白水解消化的标记蛋白质揭示，60-和35-kDa的胰凝乳蛋白酶片段的标记和标记的每一个是可重复的芪二磺酸。如果还原在中性pH下进行，则主要标记产物是对应于原始羧酸的伯醇。标记的亲和纯化条带3的酸水解产物的液相色谱显示谷氨酸而不是天冬氨酸残基已转化为羟基衍生物。这是第一次证明谷氨酸羧基在蛋白质中转化为醇。标记实验表明，有两个谷氨酸残基足够接近二苯乙烯二磺酸盐位点，以使它们的标记被4，4 '-二异硫氰基二氢二苯乙烯-2，2'-二磺酸盐和4，4 '-二硝基二苯乙烯-2，2'-二磺酸盐阻断。
A new method has been developed for the chemical modification and labeling of carboxyl groups in proteins. Carboxyl groups are activated with Woodward's reagent K (N-ethyl-5-phenylisoxazolium 3'-sulfonate), and the adducts are reduced with [3H]BH4. The method has been applied to the anion transport protein of the human red blood cell (band 3). Woodward's reagent K is a reasonably potent inhibitor of band 3-mediated anion transport; a 5-min exposure of intact cells to 2 mM reagent at pH 6.5 produces 80% inhibition of transport. The inhibition is a consequence of modification of residues that can be protected by 4,4'-dinitrostilbene-2,2'-disulfonate. Treatment of intact cells with Woodward's reagent K followed by B3H4 causes extensive labeling of band 3, with minimal labeling of intracellular proteins such as spectrin. Proteolytic digestion of the labeled protein reveals that both the 60- and the 35-kDa chymotryptic fragments are labeled and that the labeling of each is inhibitable by stilbenedisulfonate. If the reduction is performed at neutral pH the major labeled product is the primary alcohol corresponding to the original carboxylic acid. Liquid chromatography of acid hydrolysates of labeled affinity-purified band 3 shows that glutamate but not aspartate residues have been converted into the hydroxyl derivative. This is the first demonstration of the conversion of a glutamate carboxyl group to an alcohol in a protein. The labeling experiments reveal that there are two glutamate residues that are sufficiently close to the stilbenedisulfonate site for their labeling to be blocked by 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate and 4,4'-dinitrostilbene-2,2'-disulfonate.