Analysis of positional isotope exchange in ATP by cleavage of the beta P-O gamma P bond. Demonstration of negligible positional isotope exchange by myosin.
Analysis of positional isotope exchange in ATP by cleavage of the beta P-O gamma P bond. Demonstration of negligible positional isotope exchange by myosin.
复制标题
通过 β P-O γ P 键的裂解分析 ATP 中的位置同位素交换。
DOI：
10.1021/bi00399a051
复制
发表时间：
1987
期刊：
影响因子：
2.9
通讯作者：
Hackney,DD
中科院分区：
文献类型：
--
作者：
Dale,MP;Hackney,DD
卡内基梅隆大学生物科学系，匹兹堡，宾夕法尼亚州 15213 收稿日期：1987 年 4 月 20 日；修订稿于 1987 年 7 月 28 日收到摘要：提出了一种在 ATP v=* HOH 氧交换过程中分析位置同位素交换 (PIX) 的方法，该方法使用 ATP 的两步降解，导致 3-键断裂。该裂解产生源自 ATP 磷酰基的 P¡，其中包含所有四个 y 氧。 β，-桥和/3-非桥位置之间的PIX和非桥氧的冲洗可以同时通过使用在（8-非桥位置处标记有nO和在8，-桥和非桥位置处标记有lO的ATP来同时进行。将该方法应用于肌球蛋白单周转期间的ATP^HOH交换表明，在几乎不存在PIX的情况下，大部分ATP经历了非桥氧的快速冲洗。在25°C亚片段 1 的扰乱速率处于约 0. 001 s" 1 的可检测性极限，这比稳态速率慢 50 倍。这对应于对于结合 ATP 可逆水解的每个循环，扰乱结合 ADP 的 8 个氧的概率为 1 万分之一。然而，一小部分 ATP 不会经历快速冲洗。对于肌球蛋白和化学计量0°C 时的 ATP，该分数对应于 36 秒时剩余 ATP 的 10%，或初始 ATP 的 2%，并且发现在冷追实验中，ATP 的同等水平不会不可逆地与肌球蛋白结合。在抗洗脱的分数中观察到显着水平的表观 PIX，并且该表观 PIX 被证明是由于污染物腺苷酸激酶活性所致。为 Geeves 等人观察到的 PIX 提供了可能的解释。[Geeves, A., Webb, M. R., Midelfort, CF, & Trentham, DR (1980) Biochemistry 19, 4748-4754]
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213 Received April 20, 1987; Revised Manuscript Received July 28, 1987 abstract: A method for analysis of positional isotope exchange (PIX) during ATP v=* HOH oxygen exchange is presented that uses a two-step degradation of ATP resulting in cleavage of the/3-bond. This cleavage yields P¡ derived from the-phosphoryl of ATP that contains all four of the y oxygens. Both PIX between the ß,-bridge and/3-nonbridge positions and washout of the-nonbridge oxygens can be simultaneously followed by using ATP labeled with nO at the (8-nonbridge positions and lsO at the 8,-bridge andnonbridge positions. Application of this method to ATP^ HOH exchange during single turnovers of myosin indicates that the bulk of the ATP undergoes rapid washout of-nonbridge oxygens in the virtual absence of PIX. At 25 C with subfragment 1 the scrambling rate is at the limit of detectability of approximately 0. 001 s" 1, which is 50-fold slower than the steady-state rate. This corresponds to a probability of scrambling for the 8-oxygens of bound ADP of 1 in 10000 for each cycle of reversible hydrolysis of bound ATP. A fraction of the ATP, however, does notundergo rapid washout. With myosin and stoichiometric ATP at 0 C, this fraction corresponds to 10% of the ATP remaining at 36 s, or 2% of the initial ATP, and an equivalent level of ATP is found that does not bind irreversibly to myosin in a cold chase experiment. A significant level of apparent PIX is observed with subfragment 1 in the fraction that resists washout, and this apparent PIX is shown to be due to contaminant adenylate kinase activity. This apparent PIX due to adenylate kinase provides a possible explanationfor the PIX observed by Geeves et al.[Geeves,. A., Webb, M. R., Midelfort, CF, & Trentham, DR (1980) Biochemistry 19, 4748-4754] with subfragment