Direct quantitation of the number of individual penicillin-binding proteins per cell in Escherichia coli
Direct quantitation of the number of individual penicillin-binding proteins per cell in Escherichia coli
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DOI：
10.1128/jb.178.21.6110-6115.1996
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发表时间：
1996-11-01
影响因子：
3.2
通讯作者：
Pucci, MJ
中科院分区：
文献类型：
--
作者：
Dougherty, TJ;Kennedy, K;Pucci, MJ
青霉素结合蛋白（PBP）是一组参与细菌肽聚糖组装的终末阶段的酶，顾名思义，这些蛋白质也共价结合β-内酰胺抗生素并被β-内酰胺抗生素抑制。尽管许多研究已经检查了许多β-内酰胺抗生素的相对结合亲和力，令人惊讶的是，很少有研究涉及细菌细胞中存在的每种PBP的绝对数量。在本研究中，B最初报道的大肠杆菌中的PBP值大约在20年前。G. Spratt（fur.生物化学杂志72：341-352，1977）进行精制，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离来自已知数量的用[H-3]苄青霉素放射性标记的细菌的单个PBP，定位、切除放射性条带，并从凝胶切片中定量提取，通过闪烁计数测量放射性，并计算每分钟的绝对衰变，根据标记的青霉素的比活度、每分钟的绝对衰变和每毫升的CFU，确定每个细胞的每种PBP的数量。对多个样品进行测量，以对所获得的数量设置统计限制，在E.特别重要的是观察到的PBP 2和3的分子数量更高，因为已知这些PBP参与细胞形态发生。测定了丰富的Luria肉汤培养基和M9基本培养基中的PBP含量，其中基本培养基中生长较慢的细胞每个细胞具有较少的单个PBP。
The penicillin-binding proteins (PBPs) are a set of enzymes that participate in the terminal stages of bacterial peptidoglycan assembly, As their name implies, these proteins also covalently bind and are inhibited by beta-lactam antibiotics, Although many studies have examined the relative binding affinities of a number of beta-lactam antibiotics, a surprisingly small number of studies have addressed the absolute numbers of each of the PBPs present in the bacterial cell, In the present study, the PBP values initially reported in Escherichia coli almost 20 years ago by B. G. Spratt (fur. J. Biochem. 72:341-352, 1977) were refined, The individual PBPs from a known number of bacteria radiolabeled with [H-3]benzylpenicillin were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, The radioactive bands were located, excised, and quantitatively extracted from the gel slices, The radioactivity was measured by scintillation counting, and the absolute disintegrations per minute were calculated, From the specific activity of the labeled penicillin, the absolute disintegrations per minute, and the CFU per milliliter, a determination of the number of each of the PBPs per cell was made, The measurements were performed on multiple samples to place statistical limits on the numbers obtained, The values for the individual PBPs found in E. coli deviated in several ways from the previously reported observations, Of particular significance is the higher number of molecules of PBP 2 and 3 observed, since these PBPs are known to participate in cell morphogenesis. The PBP content in both rich Luria broth medium and M9 minimal medium was determined, with the slower-growing cells in minimal medium possessing fewer of the individual PBPs per cell.