EVIDENCE FOR THE 2-STEP BINDING OF ATP TO MYOSIN SUBFRAGMENT-1 BY THE RAPID-FLOW-QUENCH METHOD
EVIDENCE FOR THE 2-STEP BINDING OF ATP TO MYOSIN SUBFRAGMENT-1 BY THE RAPID-FLOW-QUENCH METHOD
复制标题
DOI：
10.1042/bj2090617
复制
发表时间：
1983-01-01
影响因子：
4.1
通讯作者：
TRAVERS, F
中科院分区：
文献类型：
--
作者：
BARMAN, TE;HILLAIRE, D;TRAVERS, F
The initial steps on the [rabbit] myosin ATPase (EC 3.6.1.3) pathway are taken to be: M + ATP .**GRAPHIC**. M .cntdot. ATP .**GRAPHIC**. M* .cntdot. ATP .**GRAPHIC**. M** .cntdot. ADP .cntdot. Pi .**GRAPHIC**. M + ADP + Pi. A 2-step binding for ATP is assumed, but the evidence for it is unconvincing: because of the rapidity of the process unambiguous values for K1 and k2 are not available. The myosin mechanism was investigated by the chemical flow-quench technique. Reaction mixtures containing [.gamma.-32P]ATP plus myosin subfragment 1 were quenched in unlabeled ATP (ATP chase) or acid (Pi burst). The ATP-chase method can lead directly to unambiguous values for K1 and k+2. The binding process was slowed down by 40% ethylene glycol. It was studied as a function of the ATP concentration. A limiting plateau resulted, showing a 2-step binding for ATP and values for K1 and k+2 were obtained. K1 and k+2 are rather sensitive to the experimental conditions. Ethylene glycol and lowering of the pH decrease both constants, but an increase in KCl concentration increases them. The binding of ATP to myosin is of an electrostatic nature. The Pi-burst method can lead directly to k+3 + k-3, but under certain conditions the kinetics are governed by K1 and k+2. This uncertainty of the interpretation of Pi-burst experiments is discussed.
