Behavior of N-phenylmaleimide- and p-phenylenedimaleimide-reacted muscle crossbridge heads.
The finding of Barnett et al. (Biophys. J. 61 (1992) 358) that NPM-reacted crossbridge heads do not bind strongly to actin in rigor solution is not easily interpreted in terms of the solution studies of Xie and Schoenberg (Biochemistry 37 (1998) 8048) who found strong binding of NPM-reacted myosin subfragment-1 to actin in solutions devoid of MgATP. For this reason, the current work uses stiffness measurement to re-investigate the binding of rabbit skeletal muscle crossbridges to actin in rigor solution. It is found that NPM-reacted crossbridge heads bind strongly to actin in rigor solution providing one is extremely careful to reduce MgATP contamination to levels well below those that would have a detectable effect on unmodified fibers. The reason for this is that NPM-reacted crossbridge heads, which hydrolyze MgATP extremely slowly, are especially susceptible to contaminant MgATP. The new fiber results show a strong correlation with the solution results. A further manifestation of this correlation is that pPDM-reacted crossbridge heads are different from NPM-reacted ones in that, like in solution, they remain weakly binding to actin even at extremely low MgATP levels. The findings suggest that the covalent crosslinking of SH1 and SH2 by pPDM is likely playing a significant role in locking pPDM-reacted crossbridge heads in a weakly binding conformation.
Li
W X
WX
Laboratory of Physical Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Schoenberg
M
M
D000199:Actins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001696:Biomechanical Phenomena; D055592:Biophysical Phenomena; D001703:Biophysics; D003432:Cross-Linking Reagents; D066298:In Vitro Techniques; D008301:Maleimides; D009119:Muscle Contraction / Q000502:physiology; D018482:Muscle, Skeletal / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D011817:Rabbits
D000199:Actins; D003432:Cross-Linking Reagents; D008301:Maleimides; D015879:Myosin Subfragments; D000255:Adenosine Triphosphate; C028525:N-phenylmaleimide; C011328:N,N'-4-phenylenedimaleimide