Probing Enzymatic Activity Inside Single Cells
| العنوان: | Probing Enzymatic Activity Inside Single Cells |
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| المؤلفون: | Aldo Jesorka, Shijun Xu, Owe Orwar, Stephen G. Weber, Ida Isaksson, Jessica Olofsson, Gavin D. M. Jeffries, Helen Bridle |
| المصدر: | Analytical Chemistry |
| بيانات النشر: | American Chemical Society (ACS), 2013. |
| سنة النشر: | 2013 |
| مصطلحات موضوعية: | Cell Membrane Permeability, 01 natural sciences, Fluorescence, Article, Analytical Chemistry, Mice, Neuroblastoma, 03 medical and health sciences, chemistry.chemical_compound, Cell Line, Tumor, Animals, Fluorescein, 030304 developmental biology, 0303 health sciences, Chromatography, Dose-Response Relationship, Drug, biology, Chemistry, 010401 analytical chemistry, Substrate (chemistry), Enzyme assay, Rats, 0104 chemical sciences, Dose–response relationship, Digitonin, Membrane, Levamisole, Biochemistry, biology.protein, Alkaline phosphatase, Single-Cell Analysis, Intracellular |
| الوصف: | We report a novel approach for determining the enzymatic activity within a single suspended cell. Using a steady-state microfluidic delivery device and timed exposure to the pore-forming agent digitonin, we controlled the plasma membrane permeation of individual NG108-15 cells. Mildly permeabilized cells (~100 pores) were exposed to a series of concentrations of fluorescein diphosphate (FDP), a fluorogenic alkaline phosphatase substrate, with and without levamisole, an alkaline phosphatase inhibitor. We generated quantitative estimates for intracellular enzyme activity and were able to construct both dose-response and dose-inhibition curves at the single-cell level, resulting in an apparent Michaelis contant Km of 15.3 μM ± 1.02 (mean ± standard error of the mean (SEM), n = 16) and an inhibition constant Ki of 0.59 mM ± 0.07 (mean ± SEM, n = 14). Enzymatic activity could be monitored just 40 s after permeabilization, and five point dose-inhibition curves could be obtained within 150 s. This rapid approach offers a new methodology for characterizing enzyme activity within single cells. |
| تدمد: | 1520-6882 0003-2700 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::bfe053d4f4b9588fcd2f998e1fb0d1e3 https://doi.org/10.1021/ac4013122 |
| حقوق: | OPEN |
| رقم الأكسشن: | edsair.doi.dedup.....bfe053d4f4b9588fcd2f998e1fb0d1e3 |
| قاعدة البيانات: | OpenAIRE |
Find this issue from the American Chemical Society | تدمد: | 15206882 00032700 |
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