Gee N S, Kenny A J
Biochem J. 1985 Sep 15;230(3):753-64. doi: 10.1042/bj2300753.
The hybridoma GK5C1, secreting a monoclonal IgG1 antibody, was generated after immunizing a mouse with pig kidney microvillar membranes. An immunoradiometric assay showed that only kidney and intestine contained detectable amounts of the antigen recognized by the antibody, the highest concentration being observed in the ileum. Immunocytochemistry confirmed this observation and revealed that the antigen was associated with renal and intestinal brush borders. By 'Western' blotting, the antigen in kidney microvilli was shown to be a 130 kDa polypeptide. Papain treatment of the membrane before blotting converted the antigen to a 125 kDa polypeptide, no longer associated with membrane. Immunoaffinity chromatography of detergent-solubilized kidney membranes yielded a pure 130 kDa protein. When one purification was monitored by the immunoradiometric assay, the yield was 3.5% and the purification factor was 1000-fold. The antigen constituted about 0.8% of the microvillar membrane protein. The protein could be reconstituted into liposomes, where electron microscopy revealed an asymmetric orientation, similar to that of ectoenzymes in this membrane. The stalk length was about 3 nm. In electron micrographs the purified protein appeared to be dimeric. A search for enzymic activity was rewarded when L-leucyl-L-tryptophan was observed to be hydrolysed. Failure to hydrolyse N-blocked peptides and the ability to release the N-terminal residue from extended peptides, including Leu-Trp-Leu and Leu-Trp-Met-Arg, showed that the activity was that of an aminopeptidase. The enzyme was maximally active at pH 7.5 and irreversibly inactivated outside the range pH 6-10. This activity could not be attributed to trace contamination with aminopeptidase N. The best substrates so far identified for the 130 kDa protein were those with tryptophan in the P1', position. This protein is a new microvillar enzyme and it is proposed that it be called aminopeptidase W.
用猪肾微绒毛膜免疫小鼠后，产生了分泌单克隆IgG1抗体的杂交瘤GK5C1。免疫放射分析表明，只有肾脏和肠道含有可检测量的该抗体识别的抗原，回肠中的浓度最高。免疫细胞化学证实了这一观察结果，并显示该抗原与肾和肠的刷状缘相关。通过“Western”印迹法，显示肾微绒毛中的抗原是一种130 kDa的多肽。印迹前用木瓜蛋白酶处理膜，可将抗原转化为125 kDa的多肽，且不再与膜结合。用去污剂溶解的肾膜进行免疫亲和层析，得到了一种纯的130 kDa蛋白。当通过免疫放射分析监测一次纯化过程时，产量为3.5%，纯化因子为1000倍。该抗原约占微绒毛膜蛋白的0.8%。该蛋白可重构到脂质体中，电子显微镜显示其具有不对称取向，类似于该膜中外切酶的取向。柄长约为3 nm。在电子显微镜照片中，纯化的蛋白似乎是二聚体。当观察到L-亮氨酰-L-色氨酸被水解时，对酶活性的搜索得到了回报。不能水解N-封闭的肽以及从包括Leu-Trp-Leu和Leu-Trp-Met-Arg在内的延伸肽中释放N-末端残基的能力，表明该活性是氨肽酶的活性。该酶在pH 7.5时活性最高，在pH 6-10范围之外不可逆地失活。这种活性不能归因于氨肽酶N的微量污染。迄今为止，为130 kDa蛋白鉴定出的最佳底物是那些在P1'位置含有色氨酸的底物。这种蛋白是一种新的微绒毛酶，建议将其称为氨肽酶W。
