Muscle cross-bridges bound to actin are disordered in the presence of 2,3-butanedione monoxime.
Electron paramagnetic resonance spectroscopy was used to monitor the orientation of muscle cross-bridges attached to actin in a low force and high stiffness state that may occur before force generation in the actomyosin cycle of interactions. 2,3-butanedione monoxime (BDM) has been shown to act as an uncompetitive inhibitor of the myosin ATPase that stabilizes a myosin.ADP.P(i) complex. Such a complex is thought to attach to actin at the beginning of the powerstroke. Addition of 25 mM BDM decreases tension by 90%, although stiffness remains high, 40-50% of control, showing that cross-bridges are attached to actin but generate little or no force. Active cross-bridge orientation was monitored via electron paramagnetic resonance spectroscopy of a maleimide spin probe rigidly attached to cys-707 (SH-1) on the myosin head. A new labeling procedure was used that showed improved specificity of labeling. In 25 mM BDM, the probes have an almost isotropic angular distribution, indicating that cross-bridges are highly disordered. We conclude that in the pre-powerstroke state stabilized by BDM, cross-bridges are attached to actin, generating little force, with a large portion of the catalytic domain of the myosin heads disordered.
Zhao
L
L
Department of Biochemistry and Biophysics, University of California, San Francisco 94143, USA.
Naber
N
N
Cooke
R
R
D000199:Actins / Q000378:metabolism*; D000205:Actomyosin / Q000378:metabolism; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D003931:Diacetyl / Q000031:analogs & derivatives* / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D066298:In Vitro Techniques; D007537:Isometric Contraction / Q000187:drug effects; D007700:Kinetics; D008433:Mathematics; D008954:Models, Biological; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D018482:Muscle, Skeletal / Q000378:metabolism / Q000502:physiology*; D009218:Myosins / Q000037:antagonists & inhibitors*; D011817:Rabbits; D013997:Time Factors
D000199:Actins; C004717:diacetylmonoxime; D000205:Actomyosin; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins; D003931:Diacetyl; D002118:Calcium