CONCURRENT ASSESSMENT OF INNER AND OUTER-MEMBRANE PERMEABILIZATION AND BACTERIOLYSIS IN ESCHERICHIA-COLI BY MULTIPLE-WAVELENGTH SPECTROPHOTOMETRY
CONCURRENT ASSESSMENT OF INNER AND OUTER-MEMBRANE PERMEABILIZATION AND BACTERIOLYSIS IN ESCHERICHIA-COLI BY MULTIPLE-WAVELENGTH SPECTROPHOTOMETRY
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DOI：
10.1016/0022-1759(88)90414-0
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发表时间：
1988-04-06
影响因子：
2.2
通讯作者：
GANZ, T
中科院分区：
文献类型：
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作者：
LEHRER, RI;BARTON, A;GANZ, T
我们开发了双波长分光光度测定法，其允许β-内酰胺酶和β-半乳糖苷酶活性可在单个样品中同时测量。我们还构建了一个靶细胞E. coli ML-35 p，其周质β-内酰胺酶和细胞质β-半乳糖苷酶，除非外膜（β-内酰胺酶）或内膜（β-半乳糖苷酶）透化。通过将分光光度法应用于整个靶细胞，我们可以确定生物制剂（包括血清、多粘菌素B和蜂毒肽）的内外膜透化动力学。通过在另外的波长下监测反应，我们还可以跟踪血清介导的细菌溶解的动力学。这些实验说明了多波长分光光度法的原理，并提供了使用它来监测和分析生物制剂对革兰氏阴性菌的作用的例子。
We developed a dual wavelength spectrophotometric assay that permitted .beta.-lactamase and .beta.-galactosidase activities to be measured concurrently in a single sample. We also constructed a target cell, E. coli ML-35p, that was substantially cryptic for its periplasmic .beta.-lactamase and cytoplasmic .beta.-galactosidase unless outer membrane (.beta.-lactamase) or inner membrane (.beta.-galactosidase) permeabilization occurred. By applying the spectrophotometric assay to whole target cells, we could ascertain the kinetics of inner and outer membrane permeabilization by biological agents, including serum, polymyxin B and mellitin. By monitoring the reaction at an additional wavelength, we could also follow the kinetics of serum-mediated bacteriolysis. These experiments illustrate the priniciple of multiple wavelength spectrophotometry and provide examples of its use to monitor and dissect the action of biological agents on a gram-negative bacterium.