Ji T H
Prog Clin Biol Res. 1976;9:171-8.
125I-Con A was reacted with the imidoester group of methyl 4-azidobenzoimidate and isolated from excess reagents on a Sephadex G-25 column. The hetero-bifunctional crosslinking reagent has two dissimilar functional groups, imidoester and arylazide, instead of two identical functional groups found in conventional homo-bifunctional crosslinking reagents. The two functional groups react under different conditions and it is possible to react one first and to activate the other later. Here the reagents were attached to the lectins by the imidoester reactions, and the arylazides now attached to the lectins were intended for the later use to crosslink the lectins and receptors. Human erythrocyte ghosts were incubated with the activated lectins, unbound lectins removed, and irradiated with UV to photolyze arylazides, thus crosslinking the lectins to the receptors in membranes. Upon the solubilization and subsequent electrophoresis of the sample, a new band appeared on the gel. 125I-Con A was detected in band A, the new band, and band 3, one of the membrane glycoproteins, diminished in parallel to the appearance of band A. The concurrent appearance of band A accompanied by 125I-Con A and decrease of band 3 were significantly reduced, when ghosts were incubated with the activated lectins but were not irradiated, ghosts were incubated with nonactivated 125I-Con A and irradiated, or ghosts were incubated with the activated concanavalin A in the presence of alpha-methylmannoside, the lectin inhibitor, and irradiated. The inhibitor removed a significant amount of the activated lectins bound to membranes, but failed to do so the activated lectins crosslinked into band A.
将125I-伴刀豆球蛋白A与4-叠氮基苯甲酸甲酯的亚氨基酯基团反应，并在Sephadex G-25柱上从过量试剂中分离出来。这种异双功能交联试剂有两个不同的官能团，即亚氨基酯和芳基叠氮，而不是传统同双功能交联试剂中发现的两个相同的官能团。这两个官能团在不同条件下反应，可以先让一个反应，然后再激活另一个。在这里，试剂通过亚氨基酯反应连接到凝集素上，现在连接到凝集素上的芳基叠氮打算稍后用于使凝集素和受体交联。将人红细胞血影与活化的凝集素一起孵育，去除未结合的凝集素，并用紫外线照射以光解芳基叠氮，从而使凝集素与膜中的受体交联。对样品进行溶解和随后的电泳后，凝胶上出现了一条新带。在A带中检测到125I-伴刀豆球蛋白A，新带出现时，膜糖蛋白之一的带3平行减少。当血影与活化的凝集素一起孵育但未照射时、血影与未活化的125I-伴刀豆球蛋白A一起孵育并照射时，或者血影在凝集素抑制剂α-甲基甘露糖苷存在下与活化的伴刀豆球蛋白A一起孵育并照射时，A带与125I-伴刀豆球蛋白A同时出现以及带3减少的情况均显著减少。该抑制剂去除了大量与膜结合的活化凝集素，但未能去除交联到A带中的活化凝集素。
