SMOOTH-MUSCLE MYOSIN - A HIGH FORCE-GENERATING MOLECULAR MOTOR

Smooth muscle generates as much force per cross sectional area of musc le as skeletal muscle with only one-fifth the myosin content. Although this apparent difference could be explained at the tissue or cellular level, it is possible that at the molecular level smooth muscle cross -bridges generate greater average force than skeletal muscle cross-bri dges. To test this hypothesis, we used an in vitro motility assay (Van Buren et al., 1994) in which either chicken thiophosphorylated gizzard smooth or pectoralis skeletal muscle monomeric myosin is adhered to a nitrocellulose surface. A fluorescently labeled actin filament, attac hed to an ultracompliant (50-200 nm/pN) glass microneedle, is brought in contact with the myosin surface. Isometric force, being generated b y myosin cross-bridges pulling on the attached actin filament, is calc ulated from the extent to which the calibrated microneedle is deflecte d. By measuring the density of myosin adhered to the surface, we estim ated the number of myosin cross-bridges that are able to interact with a length of actin filament in contact with the myosin surface. In a d irect comparison between smooth and skeletal muscle myosin, the averag e force per cross-bridge was 0.8 and 0.2 pN, respectively. Surprisingl y, smooth muscle myosin generates approximately 4 times greater averag e force per cross-bridge head than skeletal muscle myosin. Because ave rage isometric force is the product of the cross-bridge unitary force and duty cycle, we are presently using a laser optical trap in an atte mpt to measure unitary events from single myosin molecules. This appro ach should allow us to determine whether an increase in unitary force, duty cycle, or both contribute to smooth muscle myosin's enhanced for ce-generating capacity compared with skeletal muscle myosin.