Kinetics of the interaction of 2'(3')-O-(N-methylanthraniloyl)-ATP with myosin subfragment 1 and actomyosin subfragment 1: characterization of two acto-S1-ADP complexes.
We have used a fluorescent analogue of ATP, mantATP [2'(3')-O-(N-methylanthraniloyl)-adenosine 5'-triphosphate; Hiratsuka T. (1983) Biochim. Biophys. Acta 742, 496-508], and made a detailed kinetic study of the interaction of mantATP and mantADP with S1 and acto-S1. We have shown that these analogues behave like ATP and ADP, respectively. In addition, we have demonstrated that this analogue can distinguish between two acto-S1 complexes, the A-M.N (attached) and A.M.N (rigor-like) states [Geeves, M. A., Good, R. S., & Gutfreund, H. (1984) J. Muscle Res. Cell Motil. 5, 351-361]. Previously, these two states were observed with a pyrene label on Cys 374 of actin. This isomerization can now be monitored at two spatially distinct sites on the ternary complex, indicative of a major conformational change in the ternary complex. Also, we have measured the rate of ADP dissociation from both A-M.N and A.M.N directly and shown these to differ by a factor of 1000. Thus the results presented here support the model of Geeves et al. and are consistent with the A-M.N to A.M.N transition being coupled to the force-generating event of the crossbridge cycle.
Woodward
S K
SK
Division of Physical Biochemistry, National Institute for Medical Research, London, U.K.
Eccleston
J F
JF
Geeves
M A
MA
D000205:Actomyosin / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D000818:Animals; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D007700:Kinetics; D009119:Muscle Contraction; D015879:Myosin Subfragments / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D062367:ortho-Aminobenzoates / Q000378:metabolism*
D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D062367:ortho-Aminobenzoates; C066977:2'-O-(N-methylanthraniloyl)ATP; D000244:Adenosine Diphosphate; C045473:3'-O-(N-methylanthraniloyl) ATP; D000255:Adenosine Triphosphate; D000205:Actomyosin; D009218:Myosins