THE ENZYMATIC IODINATION OF THE RED CELL MEMBRANE
THE ENZYMATIC IODINATION OF THE RED CELL MEMBRANE
复制标题
红细胞膜的酶促碘化
作者：
A. Hubbard;Z. Cohn
利用乳酸过氧化物酶（LPO）、放射性碘化物和葡萄糖氧化酶-葡萄糖系统产生的过氧化氢的酶促碘化过程已被描述并用于红细胞膜的研究。97%的合并同位素在红细胞鬼影中，3%与血红蛋白有关。在没有LPO或删除任何其他试剂的情况下，红细胞膜不会发生显著的标记。超过600万倍的氯离子对碘化的抑制作用不超过50%。在单个红细胞膜中掺入多达1 × 106个碘原子不会导致显著的细胞溶解。合并标签仅在酪氨酸残基中作为单碘酪氨酸。10-15%的三氯乙酸可沉淀放射性可以用脂质溶剂提取，但以标记蛋白或125I的形式存在。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳的溶解膜蛋白显示，只有两个标记的蛋白质带的15个，每个鬼魂50-1 × 106碘原子的存在并不改变这种模式。组分a分子量为110,000，碳水化合物含量较低，占总标签的40%。组分b的表观分子量为74,000，包含所有可证明的唾液酸，占总标签的60%。经过碘化处理的完整红细胞胰蛋白酶化后，只会导致b组分消失，在培养基中出现标记的糖肽，而在膜中没有较小的标记肽。Pronase处理以类似的方式水解组分b，但也将组分a裂解为保留在膜中的72,000 mol wt的肽。蛋白酶处理和125I和131I双重标记的组合不会显示先前未暴露的蛋白质的外观。
An enzymatic iodination procedure utilizing lactoperoxidase (LPO), radioactive iodide, and hydrogen peroxide generated by a glucose oxidase-glucose system has been described and utilized for a study of the red cell membrane. 97% of the incorporated isotope is in the erythrocyte ghost and 3% is associated with hemoglobin. No significant labeling of the red cell membrane occurs in the absence of LPO or by the deletion of any of the other reagents. A 6 million-fold excess of chloride ions inhibits iodination by no more than 50%. Incorporation of up to 1 x 106 iodide atoms into a single erythrocyte membrane results in no significant cell lysis. The incorporated label is exclusively in tyrosine residues as monoiodotyrosine. 10–15% of the trichloroacetic acid-precipitable radioactivity can be extracted with lipid solvents but is present as either labeled protein or 125I. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins reveals only two labeled protein bands out of the 15 present, and the presence of 50-1 x 106 iodide atoms per ghost does not alter this pattern. Component a has a molecular weight of 110,000, is carbohydrate poor, and represents 40% of the total label. Component b has an apparent molecular weight of 74,000, contains all of the demonstrable sialic acid, and accounts for 60% of the total label. Trypsinization of iodinated, intact red cells results in the disappearance of only component b, the appearance of labeled glycopeptides in the medium, and the absence of smaller, labeled peptides remaining in the membrane. Pronase treatment hydrolyzes component b in a similar fashion, but also cleaves component a to a 72,000 mol wt peptide which is retained in the membrane. A combination of protease treatment and double labeling with 125I and 131I does not reveal the appearance of previously unexposed proteins.