Good A H, Craik J D, Jarvis S M, Kwong F Y, Young J D, Paterson A R, Cass C E
McEachern Laboratory, University of Alberta, Edmonton, Canada.
Biochem J. 1987 Jun 15;244(3):749-55. doi: 10.1042/bj2440749.
Three monoclonal antibodies have been raised against partially purified band 4.5 polypeptides [Steck (1974) J. Cell Biol. 62, 1-19] from pig erythrocyte membranes. The antibodies were capable of binding to both intact pig erythrocytes and protein-depleted membrane preparations and recognized detergent-solubilized polypeptides from adult and neonatal pig erythrocytes that were photolabelled with [G-3H]nitrobenzylthioinosine (NBMPR), a potent specific inhibitor of nucleoside transport. The antibodies did not recognize polypeptides from neonatal pig erythrocytes that were photolabelled with the glucose-transport inhibitor [3H]cytochalasin B. Reactivity with polypeptides of apparent Mr 64,000 [10% (w/v) acrylamide gels] was demonstrated by Western-blot analysis. The antibodies recognized pig band 4.5 polypeptides after prolonged treatment with endoglycosidase F, a finding consistent with reactivity against polypeptide, rather than carbohydrate, determinants. Trypsin digestion of NBMPR-labelled protein-depleted pig erythrocyte membranes generated two labelled polypeptide fragments (Mr 43,000 and 26,000). Two of the antibodies recognized both fragments on Western blots, whereas the third bound to the larger, but not to the smaller, fragment. The antibodies had no significant effect on reversible binding of NBMPR to protein-depleted pig erythrocyte membranes and did not bind to NBMPR-labelled polypeptides in human, rabbit or mouse erythrocytes.
已针对从猪红细胞膜中部分纯化的4.5带多肽[Steck（1974年），《细胞生物学杂志》62卷，1 - 19页]制备了三种单克隆抗体。这些抗体能够与完整的猪红细胞以及去除蛋白质的膜制剂结合，并识别来自成年和新生猪红细胞的经去污剂溶解的多肽，这些多肽用[G - 3H]硝基苄硫肌苷（NBMPR）进行光标记，NBMPR是一种有效的核苷转运特异性抑制剂。这些抗体不能识别用葡萄糖转运抑制剂[3H]细胞松弛素B进行光标记的新生猪红细胞的多肽。通过蛋白质印迹分析证明了与表观分子量为64,000的多肽（在10%（w/v）丙烯酰胺凝胶上）有反应性。在用内切糖苷酶F长时间处理后，这些抗体识别猪4.5带多肽，这一发现与针对多肽而非碳水化合物决定簇的反应性一致。用胰蛋白酶消化经NBMPR标记的去除蛋白质的猪红细胞膜产生了两个标记的多肽片段（分子量分别为43,000和26,000）。其中两种抗体在蛋白质印迹上识别这两个片段，而第三种抗体只与较大的片段结合，不与较小的片段结合。这些抗体对NBMPR与去除蛋白质的猪红细胞膜的可逆结合没有显著影响，并且不与人、兔或小鼠红细胞中经NBMPR标记的多肽结合。
