Biophysical Journal, March 2000, p. 1474-1481,
Vol. 78, No. 3
CaATP as a Substrate to Investigate the Myosin Lever Arm Hypothesis of Force Generation
*Department of Biochemistry,
University of the Pacific, School of Dentistry, San Francisco, California
94115-2399; In an effort to test the lever arm model of force generation, the effects of replacing magnesium with calcium as the ATP-chelated divalent cation were determined for several myosin and actomyosin reactions. The isometric force produced by glycerinated muscle fibers when CaATP is the substrate is 20% of the value obtained with MgATP. For myosin subfragment 1 (S1), the degree of lever arm rotation, determined using transient electric birefringence to measure rates of rotational Brownian motion in solution, is not significantly changed when calcium replaces magnesium in an S1-ADP-vanadate complex. Actin activates S1 CaATPase activity, although less than it does MgATPase activity. The increase in actin affinity when S1 · CaADP · Pi is converted to S1 · CaADP is somewhat greater than it is for the magnesium case. The ionic strength dependence of actin binding indicates that the change in apparent electrostatic charge at the acto-S1 interface for the S1 · CaADP · Pi to S1 · CaADP step is similar to the change when magnesium is bound. In general, CaATP is an inferior substrate compared to MgATP, but all the data are consistent with force production by a lever arm mechanism for both substrates. Possible reasons for the reduced magnitude of force when CaATP is the substrate are discussed. |
Department
of Chemistry and Biochemistry, San Francisco State University, San Francisco,
California 94132; and 
Cardiovascular
Research Institute, University of California, San Francisco, California
94143 USA