Fluorescent coumarin-labeled nucleotides to measure ADP release from actomyosin
Fluorescent coumarin-labeled nucleotides to measure ADP release from actomyosin
复制标题
DOI：
10.1016/s0006-3495(01)75810-9
复制
发表时间：
2001-09-01
影响因子：
3.4
通讯作者：
Corrie, JET
中科院分区：
文献类型：
--
作者：
Webb, MR;Corrie, JET
基于2 '（3 '）-O-（2-氨乙基）氨甲酰基-ATP（edaATP），已经合成了几种香豆素标记的核苷酸。与游离氨基偶联的荧光香豆素是7-二乙基氨基香豆素-3-羧酸（以产生deac-edaATP）、香豆素343（but-edaATP）和7-乙基氨基-8-溴香豆素-3-羧酸（mbc-edaATP）。这些核苷酸类似物的氨基甲酰基键非常缓慢地经历2 '-和3 ' -羟基连接之间的相互转化，使得2 '-和3 ' -异构体分离并以最小平衡储存。3 ' -Deac-edaADP在430 nm和477 nm处具有荧光激发和发射最大值，荧光量子产率为0.012。3 ' -but-edaADP的等效数据分别为445 nm、494 nm和0.51，3 ' -mbc-edaADP的等效数据分别为405 nm、464 nm和0.62。与骨骼肌球蛋白亚片段1的相互作用进行了测量，在存在和不存在的肌动蛋白。在每种情况下，当与亚片段1结合时，荧光降低，3 ' -deac-edaADP降低3倍，3 ' -but-edaADP降低7倍，3 ' -mbc-edaADP降低11倍。稳态ATP酶的测量和动力学的结合和释放的核苷酸是类似的天然核苷酸的报告。大的荧光变化，可以观察到这些类似物从肌动球蛋白亚片段1的释放，使这个过程的动力学的直接测量。在3 ' -deac-edaADP的情况下，测得的速率常数为474 s（-1）（在pH 7.0、20 ℃和低离子强度下）。
Several coumarin-labeled nucleotides have been synthesized, based on 2 ' (3 ')-O-(2-aminoethyl)carbamoyl-ATP (edaATP). The fluorescent coumarins coupled with the free amino group are 7-diethylaminocoumarin-3-carboxylic acid (to give deac-edaATP), coumarin 343 (but-edaATP) and 7-ethylamino-8-bromocoumarin-3-carboxylic acid (mbc-edaATP). The carbamoyl linkage of these nucleotide analogs undergoes interconversion between 2 '- and 3 ' -hydroxyl attachment very slowly, so that the 2 '- and 3 ' -isomers were separated and stored with minimal equilibration. 3 ' -Deac-edaADP had fluorescence excitation and emission maxima at 430 nm and 477 nm, with a fluorescence quantum yield of 0.012. The equivalent data for 3 ' -but-edaADP are 445 nm, 494 nm, and 0.51, respectively, and for 3 ' -mbc-edaADP, 405 nm, 464 nm, and 0.62. The interaction with skeletal myosin subfragment 1 was measured in the absence and presence of actin. In each case the fluorescence was decreased when bound to subfragment 1, 3-fold for 3 ' -deac-edaADP, 7-fold for 3 ' -but-edaADP, and 11-fold for 3 ' -mbc-edaADP. Steady-state ATPase measurements and the kinetics of binding and release of nucleotides were similar to those reported for the natural nucleotide. Large fluorescence changes could be observed for the release of these analogs from actomyosin subfragment 1, enabling a direct measurement of the kinetics of this process. In the case of 3 ' -deac-edaADP a rate constant of 474 s(-1) was measured (at pH 7.0, 20 degreesC, and low ionic strength).