Xu Y, Barbieri J T
Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226.
Infect Immun. 1995 Mar;63(3):825-32. doi: 10.1128/iai.63.3.825-832.1995.
Pertussis toxin (PT)-catalyzed ADP-ribosylation of target proteins in intact Chinese hamster ovary (CHO) cells was evaluated with an in vitro ADP-ribosylation assay. In this assay, a postnuclear supernatant was prepared from CHO cells and used as a source of PT-sensitive target proteins for in vitro [32P[ADP-ribosylation. The postnuclear supernatant contained three proteins that were ADP-ribosylated in vitro, with apparent molecular masses of 50, 45, and 42 kDa. The 42- and 45-kDa proteins were membrane associated, while the 50-kDa protein was soluble. Following PT treatment of CHO cells, the 42- and 45-kDa proteins were not available for in vitro ADP-ribosylation, while the soluble 50-kDa protein remained available for in vitro ADP-ribosylation. The decrease in the availability of the 42- and 45-kDa proteins to in vitro ADP-ribosylation was proportional to the PT concentration and time of incubation with CHO cells. Western immunoblot analysis showed that extracts from PT-treated CHO cells and control CHO cells possessed equivalent amounts of two proteins that were recognized by anti-Gi protein antiserum. The two proteins recognized by anti-Gi protein antiserum from PT-treated cells migrated with higher apparent molecular weights than the two proteins from control cells. This was consistent with the in vivo ADP-ribosylation of the two proteins by PT.(ABSTRACT TRUNCATED AT 250 WORDS)
通过体外ADP-核糖基化试验评估了百日咳毒素（PT）对完整的中国仓鼠卵巢（CHO）细胞中靶蛋白的催化ADP-核糖基化作用。在该试验中，从CHO细胞制备核后上清液，并将其用作体外[32P]ADP-核糖基化的PT敏感靶蛋白来源。核后上清液包含三种在体外被ADP-核糖基化的蛋白，其表观分子量分别为50、45和42 kDa。42 kDa和45 kDa的蛋白与膜相关，而50 kDa的蛋白是可溶性的。用PT处理CHO细胞后，42 kDa和45 kDa的蛋白不能用于体外ADP-核糖基化，而可溶性50 kDa的蛋白仍可用于体外ADP-核糖基化。42 kDa和45 kDa蛋白用于体外ADP-核糖基化的可用性降低与PT浓度和与CHO细胞孵育的时间成正比。蛋白质免疫印迹分析表明，PT处理的CHO细胞和对照CHO细胞的提取物中具有等量的两种可被抗Gi蛋白抗血清识别的蛋白。PT处理细胞的抗Gi蛋白抗血清识别的两种蛋白迁移时的表观分子量高于对照细胞的两种蛋白。这与PT对这两种蛋白的体内ADP-核糖基化作用一致。（摘要截短于250字）
