Hara Kiyotaka Y, Mori Hideo
Biofrontier Laboratories Kyowa Hakko Kogyo Co. Ltd. 3-6-6 Asahimachi Machidashi, Tokyo 194-8533, Japan.
J Biomol Screen. 2006 Apr;11(3):310-7. doi: 10.1177/1087057105285112. Epub 2006 Feb 20.
The authors have developed an efficient method to measure cellular activity of ATP synthesis. Although ATP is a major energy source of biological reactions, it has been difficult to measure cellular ATP synthetic activity quantitatively. In this report, bioluminescence from the luciferin-luciferase reaction was used for the quantitative measurement. Under the used condition, bioluminescence from standard ATP solution showed no attenuation within several minutes, and the intensity corresponded proportionally to ATP concentrations of the standards. To measure dynamic cellular ATP synthetic activity, combination of osmotic shock and detergent treatment was used to make Escherichia coli cells permeable. ATP was discharged from permeable cells and reacted with externally added luciferase. Because permeable cells used glucose to synthesize and accumulate ATP without further growth, intensity of bioluminescence was increasing during the cellular consumption of glucose. Cellular ATP biosynthetic activity was calculated form the slope of linearly increasing bioluminescence. This permeable cell assay could be applied to high-throughput measuring for dynamic cellular activity of glycolytic ATP synthesis.
作者开发了一种高效的方法来测量细胞中ATP合成的活性。尽管ATP是生物反应的主要能量来源，但一直难以定量测量细胞的ATP合成活性。在本报告中，荧光素-荧光素酶反应产生的生物发光被用于定量测量。在所使用的条件下，标准ATP溶液产生的生物发光在几分钟内没有衰减，并且强度与标准品的ATP浓度成比例。为了测量细胞ATP合成的动态活性，采用渗透压休克和去污剂处理相结合的方法使大肠杆菌细胞具有通透性。ATP从通透细胞中释放出来，并与外部添加的荧光素酶发生反应。由于通透细胞利用葡萄糖合成并积累ATP而不再进一步生长，因此在细胞消耗葡萄糖的过程中生物发光强度不断增加。细胞ATP生物合成活性通过生物发光线性增加的斜率来计算。这种通透细胞测定法可应用于糖酵解ATP合成的细胞动态活性的高通量测量。
