Abstract. Although it is generally believed that phosphorylation of the regulatory light chain of myosin is required before smooth muscle can develop force, it is not known if the overall degree of phosphorylation can also modulate the rate at which cross-bridges cycle. To address this question, an in vitro motility assay was used to observe the motion of single actin filaments interacting with smooth muscle myosin copolymers composed of varying ratios of phosphorylated and unphosphorylated myosin. The results suggest that unphosphorylated myosin acts as a load to slow down the rate at which actin is moved by the faster cycling phosphorylated cross-bridges. Myosin that was chemically modified to generate a noncycling analogue of the "weakly" bound conformation was similarly able to slow down phosphorylated myosin. The observed modulation of actin velocity as a function of copolymer composition can be accounted for by a model based on mechanical interactions between cross-bridges.

The double-headed myosin V molecular motor carries intracellular cargo processively along actin tracks in a hand-over-hand manner. To test this hypothesis at the molecular level, we observed single myosin V molecules that were differentially labeled with quantum dots having different emission spectra so that the position of each head could be identified with approximately 6-nm resolution in a total internal reflectance microscope. With this approach, the individual heads of a single myosin V molecule were observed taking 72-nm steps as they alternated positions on the actin filament during processive movement. In addition, the heads were separated by 36 nm during pauses in motion, suggesting attachment to actin along its helical repeat. The 36-nm interhead spacing, the 72-nm step size, and the observation that heads alternate between leading and trailing positions on actin are obvious predictions of the hand-over-hand model, thus confirming myosin V's mode of walking along an actin filament.

Differences in the mechanical properties of mammalian smooth, skeletal, and cardiac muscle have led to the proposal that the myosin isozymes expressed by these tissues may differ in their molecular mechanics. To test this hypothesis, mixtures of fast skeletal, V1 cardiac, V3 cardiac and smooth muscle (phosphorylated and unphosphorylated) myosin were studied in an in vitro motility assay in which fluorescently-labelled actin filaments are observed moving over a myosin coated surface. Pure populations of each myosin produced actin filament velocities proportional to their actin-activated ATPase rates. Mixtures of two myosin species produced actin filament velocities between those of the faster and slower myosin alone. However, the shapes of the myosin mixture curves depended upon the types of myosins present. Analysis of myosin mixtures data suggest that: (1) the two myosins in the mixture interact mechanically and (2) the same force-velocity relationship describes a myosin's ability to operate over both positive and negative forces. These data also allow us to rank order the myosins by their average force per cross-bridge and ability to resist motion (phosphorylated smooth > skeletal = V3 cardiac > V1 cardiac). The results of our study may reflect the mechanical consequence of multiple myosin isozyme expression in a single muscle cell.

To determine whether the apparent enhanced force-generating capabilities of smooth muscle relative to skeletal muscle are inherent to the myosin cross-bridge, the isometric steady-state force produced by myosin in the in vitro motility assay was measured. In this assay, myosin adhered to a glass surface pulls on an actin filament that is attached to an ultracompliant nm/pN) glass microneedle. The number of myosin cross-bridge heads able to interact with a length of actin filament was estimated by measuring the density of biochemically active myosin adhered to the surface; with this estimate, the average force per cross-bridge head ofsmooth and skeletal muscle myosins is 0.6 pN and 0.2 pN, respectively. Surprisingly, smooth muscle myosin generates approximately three times greater average force per cross-bridge head than does skeletal muscle myosin.All muscles are believed to generate force through the cyclic interaction of two contractile proteins, myosin and actin. The energy that drives this process is derived from the hydrolysis of ATP by the myosin cross-bridge. Smooth muscle, found in virtually all hollow organs of the body, is unique in its ability to generate as much force per cross-sectional area as skeletal muscle with only one-fifth the myosin content (1). Although this apparent difference could possibly be explained at the tissue or cellular level, when these data are interpreted at the molecular level, smooth muscle cross-bridges may generate greater average force than skeletal muscle cross-bridges. To test this hypothesis, we used an in vitro motility assay.The in vitro motility assay has proven a useful tool in studying the molecular mechanism by which myosin generates force and motion as it interacts with actin (2-4). In this assay, fluorescently labeled actin flaments are observed sliding over a myosin-coated surface. Although the velocity of freely moving actin filaments can be readily determined, the measurement of force is far more difficult. In 1988, Kishino and Yanagida (5) described a novel technique for measuring force in this assay. We have adopted this technique ( Fig. 1) to compare the force generated by smooth and skeletal muscle myosin. MATERIALS AND METHODSAll contractile proteins were prepared as described (4). Either turkey gizzard thiophosphorylated smooth muscle or chicken pectoralis skeletal muscle myosin monomers (250 ,g/ml) in a myosin buffer (300 mM KCl/5 mM MgCl2/25 mM imidazole/1 mM EGTA/10 mM dithiothreitol, pH 7.4) were incubated on a nitrocellulose-coated glass coverslip for 2 min and then washed with bovine serum albumin at 0.5 mg/ml in myosin buffer. Next, a 0.1-ml-solution bead of low-ionic-strength assay buffer containing fluorescent, tetramethylrhodamine B isothiocyanate-labeled actin filaments (-45 0.375% methylcellulose, pH 7.4, with an enzymatic oxygen scavenger system (glucose oxidase at 0.1 mg/ml, catalase at 0.018 mg/ml, and glucose at 2.3 mg/ml)].Actin filaments (10-30 ,um in length), suspended in solution and freely moving on the myosin surface, w...