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Transient kinetic methods were used to study interactions between actin, MgADP, and smooth muscle (chicken gizzard) myosin subfragment 1 (smS1). The equilibrium dissociation constant (Kd) of actin for smS1 was 3.5 nM, tighter than that of skeletal S1 (skS1). Actin binding to smS1 was weakened 5-fold by saturation with ADP compared to 30-60-fold for skS1.(More)
Kinesin-1 is a molecular motor protein that transports cargo along microtubules. Inside cells, the vast majority of kinesin-1 is regulated to conserve ATP and to ensure its proper intracellular distribution and coordination with other molecular motors. Regulated kinesin-1 folds in half at a hinge in its coiled-coil stalk. Interactions between coiled-coil(More)
To understand the domain requirements of phosphorylation-dependent regulation, we prepared three recombinant constructs of nonmuscle heavy meromyosin IIB containing 1) two complete heads, 2) one complete head and one head lacking the motor domain, and 3) one complete head and one head lacking both motor and regulatory domains. Steady-state ATPase(More)
Ultraviolet irradiation above 300 nm of the stable MgADP-orthovanadate (Vi)-myosin subfragment 1 (S1) complex resulted in covalent modification of the S1 and in the rapid release of trapped MgADP and Vi. This photomodified S1 had Ca2+ATPase activity 4-5-fold higher than that of the non-irradiated control S1, while the K+EDTA-ATPase activity was below 10% of(More)
Recent structural evidence (Rayment, I., Holden, H. M., Whittaker, M., Yohn, C. B., Lorenz, M., Holmes, K. C., and Milligan, R. A. (1993) Science 261, 58-65) suggests that the two heads of skeletal muscle myosin interact when the protein is bound to filamentous actin. Direct chemical cross-linking experiments show that the two heads of smooth muscle myosin(More)
Reactions involving proteins frequently involve large changes in volume, which allows the equilibrium position to be perturbed by changes in pressure. Rapid changes in pressure can thus be used to initiate relaxation in pressure; however, this approach is seldom used, because it requires specialized equipment. We have built a microvolume (50 microl)(More)
Current theories of muscle cross-bridge function suggest that force is generated by a change in the orientation of the myosin neck region. We attached a paramagnetic probe to a subunit in the neck region and measured the orientation of the probe using electron paramagnetic resonance spectroscopy. The angle of the probes on smooth myosin S1 were changed by(More)
Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) is characterized by a serine-threonine kinase domain, an autoinhibitory domain, a calmodulin-binding domain and a neural visinin-like domain with three EF-hands. The neural visinin-like Ca(2+)-binding domain at the C-terminal end of the CaM-binding domain makes CCaMK unique among all the known(More)
Smooth myosin regulatory light chain (RLC) was exchanged with RLC labeled with benzophenone-4-iodoacetamide at Cys-108. Irradiation under conditions that favor the folded (10 S) conformation resulted in 10 S cross-linked myosin that could not unfold. Purified 10 S cross-linked myosin was cross-linked between the RLC of one head to light meromyosin between(More)
Myosin subfragment 1 (S1) can be specifically photomodified at the active site without polypeptide chain cleavage by irradiating the stable MgADP-orthovanadate-S1 complex with UV light above 300 nm [Grammer, J. C., Cremo, C. R., & Yount, R. G. (1988) Biochemistry (preceding paper in this issue)]. Here, the UV spectral properties of photomodified S1 were(More)