Abstract
The Nitella-based in vitro motility assay developed by Sheetz and Spudich (Sheetz, M.P., and Spudich, J. A. (1983) Nature 303, 31-35) is a quantitative assay for measuring the velocity of myosin-coated beads over an organized substratum of actin. We have used this assay to analyze the effect of phosphorylation of various sites on the 20,000-Da light chain of smooth muscle and cytoplasmic myosins. Phosphorylation by myosin light chain kinase at serine 19 on the 20,000-Da light chain subunit of smooth muscle myosin from turkey gizzard, bovine trachea and aorta, and of cytoplasmic myosin from human platelets was required for bead movement. The individual phosphorylated myosin-coated beads moved at characteristic rates under the same conditions (turkey gizzard myosin, 0.2 micron/s; aorta or trachea myosin, 0.12 micron/s; and platelet myosin, 0.04 micron/s; in contrast, rabbit skeletal muscle myosin, 2 micron/s). Myosin light chain kinase can also phosphorylate threonine 18 in addition to serine 19, and this phosphorylation resulted in an increase in the actin-activated MgATPase activity (Ikebe, M., and Hartshorne, D.J. (1985) J. Biol. Chem. 260, 10027-10031). Phosphorylation at this site had no effect on the velocity of smooth muscle myosin-coated beads. Protein kinase C (Ca2+/phospholipid-dependent enzyme) can also phosphorylate two to three sites on the 20,000-Da light chain, and this phosphorylation alone did not result in the movement of myosin-coated beads. When myosin that had been previously phosphorylated by myosin light chain kinase at serine 19 was also phosphorylated by protein kinase C, myosin-coated beads moved at the same velocity as beads coated with myosin phosphorylated by myosin light chain kinase alone. Tropomyosin binding to actin also had an activating effect on the actin-activated MgATPase activity through an effect on the Vmax and also resulted in an increase in the velocity of myosin-coated beads.
Highlights
The Nitella-based in vitro motility assay developed and the preferred residue is serine 19 on the 20,000-Da light by Sheetz and Spudich
Myosinlight chain kinase canalso phos- ation of myosin by protein kinase C alone has little or no phorylate threonine 18 in addition to serine 19, and effect on the actin-activated MgATPase activity, whereas this phosphorylation resultedin an increase in the phosphorylation of smooth muscle myosin by protein kinase actin-activated MgATPase activity
When myosinthat had been previously phosphorylated bymyosin light chain kinase at serine 19 was phosphorylated by proteinkinase C, myosin-coated beads moved at the same velocity as beads coated with myosin phosphorylated by myosin light chain kinase alone
Summary
[ethylenebis(oxyethylenenitrilo)]tetraaceticacid; MOPS, 4-morpholinepropanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; of smooth muscle and cytoplasmic myosin-coated beads in the Nitella-based in vitro motility assay and compare the results of this assay with those of the more conventional steady-state kinetic analysis of the actin-activated MgATPase activity. We examine the effect of tropomyosin on HMM, heavy meromyosin. The movement of myosin-coated beads and on the kinetics of Smooth Muscle and Cytoplasmic Myosin-coated Bead Movement actin-myosininteraction.Some of this material has been min. The pellet was dissolved in 25 mM NaCl, 10 mM MgCl,, 10 mM presented in preliminary form [22]. MOPS (pH 7.0), 1 mM dithiothreitol, 3 mM NaN3, 0.1 mM PMSF, 5 mg/liter leupeptin and dialyzed against the same solution overnight
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