TOPOLOGY OF PENICILLIN-BINDING PROTEIN-1B OF ESCHERICHIA-COLI AND TOPOGRAPHY OF 4 ANTIGENIC DETERMINANTS STUDIED BY IMMUNOCOLABELING ELECTRON-MICROSCOPY
TOPOLOGY OF PENICILLIN-BINDING PROTEIN-1B OF ESCHERICHIA-COLI AND TOPOGRAPHY OF 4 ANTIGENIC DETERMINANTS STUDIED BY IMMUNOCOLABELING ELECTRON-MICROSCOPY
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DOI：
10.1128/jb.172.1.71-79.1990
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发表时间：
1990-01-01
影响因子：
3.2
通讯作者：
NANNINGA, N
中科院分区：
文献类型：
--
作者：
DENBLAAUWEN, T;NANNINGA, N
建立了一种研究蛋白质在大肠杆菌细胞质膜上定位的方法。将来自超声处理细胞的囊泡在电子显微镜支持网格上的液滴中依次与针对具有未知方向的蛋白质（PBP 1b）的单克隆抗体（MAb）孵育，然后与针对周质组分（肽聚糖）的MAb孵育。分别用5-和10-nm金缀合的二抗使不同的MAb可见。PBP 1b与肽聚糖共标记。用针对四种不同表位的单克隆抗体标记膜囊泡中的PBP 1b，进一步用于估计每个细胞中PBP 1b分子的数量。在肉汤中生长的每个细胞约标记1，400个PBP 1b分子。通过单克隆抗体对的免疫共标记和竞争性抗体结合抑制来研究单克隆抗体的表位的空间分布。初步推断，这4个表位形成了一个抗原决定簇，占据了不到一半的PBP 1b表面。
A method has been developed to study the orientation of proteins in the cytoplasmic membrane of Escherichia coli. Vesicles from sonicated cells were incubated in droplets on electron microscope support grids in sequence with a monoclonal antibody (MAb) against a protein with an unknown orientation (PBP 1b) followed by a MAb against a periplasmic component (peptidoglycan). The different MAbs were made visible with 5- and 10-nm gold-conjugated secondary antibodies, respectively. PBP 1b appeared to colabel with peptidoglycan. The labeling of PBP 1b in membrane vesicles with MAbs against four different epitopes was further used to estimate the number of PBP 1b molecules per cell. Approximately 1,400 PBP 1b molecules per cell grown in broth were labeled. The spatial distribution of the epitopes of the MAbs was studied by immunocolabeling of pairs of MAbs and by competitive antibody-binding inhibition. It could be tentatively concluded that the four epitopes form a cluster of antigenic determinants which occupy less than half of the surface of PBP 1b.