Preferential binding of the neutrophil cytoplasmic granule-derived bactericidal/permeability increasing protein to target bacteria. Implications and use as a means of purification.
Preferential binding of the neutrophil cytoplasmic granule-derived bactericidal/permeability increasing protein to target bacteria. Implications and use as a means of purification.
复制标题
DOI：
10.4049/jimmunol.142.8.2807
复制
发表时间：
1989-04
影响因子：
4.4
通讯作者：
B. Mannion;E. Kalatzis;Jerrold Weiss;P. Elsbach
中科院分区：
文献类型：
--
作者：
B. Mannion;E. Kalatzis;Jerrold Weiss;P. Elsbach
中性粒细胞(PMN)的碱性杀菌/通透性增加蛋白(BPI)对革兰氏阴性细菌的特异性归因于它对负电荷包膜内毒素的强烈吸引力。PMN匀浆或提取物对大肠杆菌的抗菌活性与其BPI含量相对应，并被抗BPI抗体阻断，表明BPI的作用不受其他PMN蛋白的影响。为了测试当存在其他抗菌蛋白时，BPI是否优先与大肠杆菌结合，我们测量了在存在和不存在其他人PMN颗粒蛋白的情况下，[125I]人BPI在缓冲液(pH 7.5)平衡盐溶液中与E.ColiJ5的结合。BPI结合是饱和的，表观K=23 nm，结合部位数为220万个/细胞。人和兔的BPI竞争性抑制人BPI的结合，而髓过氧化物酶、溶菌酶和组织蛋白酶G仅弱抑制人BPI与[125I]的结合。此外，与中性粒细胞或慢性粒细胞白血病脾的粗提物孵育后，经洗涤的大肠杆菌颗粒的银染和免疫印迹表明，BPI与BPI几乎是定量结合的，而与其他白细胞蛋白没有明显的结合(添加的总蛋白的98%以上在上清液中回收)。加入200 mM的氯化镁后，结合的BPI中约有80%以完全活性和纯净的形式释放出来(通过SDS-PAGE和HPLC法判断)。因此，PMN粗提物中的BPI选择性和可逆地结合到靶细菌上，提供了一步“亲和”纯化过程。
The specificity of the basic bactericidal/permeability increasing protein (BPI) of polymorphonuclear leukocytes (PMN) for gram-negative bacteria is attributable to its strong attraction for the negatively charged envelope LPS. The antibacterial activity of PMN homogenates or extracts toward Escherichia coli corresponds to their BPI content and is blocked by anti-BPI IgG, suggesting that BPI action is unaffected by the presence of other PMN proteins. To test if BPI is preferentially bound to E. coli when other antibacterial proteins are present, we have measured binding in buffered (pH 7.5) balanced salts solution of [125I] human BPI to E. coli J5 in the presence and absence of other human PMN granule proteins. BPI binding is saturable with an apparent K = 23 nM and 2.2 million binding sites/cell. While binding of [125I] human BPI is competitively inhibited by human or rabbit BPI, it is only weakly inhibited by myeloperoxidase, lysozyme, or cathepsin G. In contrast, myeloperoxidase binding to E. coli is strongly inhibited by BPI. Moreover, incubation of E. coli with crude extracts of PMN or CML spleen results in near quantitative binding of BPI, identified by silver staining and immunoblotting after SDS-PAGE of the washed E. coli pellet, without recognizable binding of other leukocyte proteins (greater than 98% of added total protein is recovered in supernatant). After addition of 200 mM MgCl2, approximately 80% of bound BPI is released as fully active and pure protein (as judged by SDS-PAGE and HPLC). Thus the selective and reversible binding of BPI in crude PMN extracts to target bacteria provides a one-step "affinity" purification procedure.