Perforin is a 68 kD protein found in the granules of cytotoxic lymphocytes and is used by lymphocytes to form lethal pores in the membranes of the cells they kill. We and others have found that when perforin is purified, its lytic activity is markedly reduced. ELISAs indicated that our final recovery of perforin protein was excellent. We decided to determine if depletion of other granule proteins contributed to the loss of lytic activity. We isolated perforin to the point where lytic activity was diminished and added back granule proteins that had no lytic activity or detectable (antigenic) perforin. Perforin was isolated by Cu2+-immobilized metal affinity chromatography (IMAC) followed by phenyl-Superose hydrophobic interaction chromatography (HIC). Its lytic activity was enhanced by a low molecular weight (<15 kD) protein, perforin enhancing protein (PEPr). We have isolated PEPr by two methods, HIC and MonoQ. Nonlytic PEPr restored perforin to close to its original lytic activity. A protein similar if not identical to PEPr was also detectable as an 125I-labeled protein associated with lytic perforin. We propose that PEPr acts in conjunction with perforin to form lethal pores and suggest that PEPr may be the rat equivalent of the human cytotoxic lymphocyte protein, granulysin.

译文

穿孔素是一种68 kD蛋白,存在于细胞毒性淋巴细胞的颗粒中,被淋巴细胞用来在其杀死的细胞膜上形成致死孔。我们和其他人已经发现,当穿孔素被纯化时,其裂解活性显着降低。 ELISA法表明,我们穿孔蛋白的最终回收率极高。我们决定确定其他颗粒蛋白的消耗是否导致裂解活性降低。我们将穿孔素分离到溶解活性降低的位置,并添加没有溶解活性或可检测(抗原性)穿孔素的颗粒蛋白。通过铜固定化金属亲和色谱法(IMAC),然后进行苯基-Superose疏水相互作用色谱法(HIC)分离穿孔素。低分子量(<15 kD)蛋白,穿孔素增强蛋白(PEPr)增强了其裂解活性。我们通过两种方法HIC和MonoQ分离了PEPr。非溶解性PEPr使穿孔素恢复至接近其原始溶解活性。与PEPr相似但不相同的蛋白质也可以作为与裂解穿孔素相关的125 I标记蛋白质检测出来。我们建议PEPr与穿孔素一起形成致死性毛孔,并建议PEPr可能与人细胞毒性淋巴细胞蛋白颗粒溶素相当。

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