BEHAVIOR OF N-PHENYLMALEIMIDE-REACTED AND P-PHENYLENEDIMALEIMIDE-REACTED MUSCLE CROSSBRIDGE HEADS

The finding of Barnett et al. (Biophys. J. 61 (1992) 358) that NPM-rea cted 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-l to actin in solutions devoid of MgATP . For this reason, the current work uses stiffness measurement to re-i nvestigate the binding of rabbit skeletal muscle crossbridges to actin in rigor solution. It is found that NPM-reacted crossbridge heads bin d strongly to actin in rigor solution providing one is extremely caref ul 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 s lowly, 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 he ads are different from NPM-reacted ones in that, like in solution, the y remain weakly binding to actin even at extremely low MgATP levels. T he findings suggest that the covalent crosslinking of SH1 and SH2 by p PDM is likely playing a significant role in locking pPDM-reacted cross bridge heads in a weakly binding conformation. (C) 1998 Published by E lsevier Science B.V. All rights reserved.