In vitro motilities of the unconventional myosins, brush border myosin-I, and chick brain myosin-V exhibit assay-dependent differences in velocity.
Two types of in vitro motility assays are currently used for examining the mechanochemical properties of purified myosins. The Nitella bead movement assay (Sheetz and Spudich: Nature 303:31-35, 1983) allows determination of both velocity and directionality of movement, but is of limited utility because of the fragile nature of the dissected Nitella internodal cells. On the other hand, the sliding actin filament assay (Kron and Spudich: Proc. Natl. Acad. Sci. U.S.A. 83:6272-6276, 1986) is technically much simpler to perform than the Nitella assay, and is suitable for the study of numerous physiological parameters. As it is currently used, however, the sliding actin filament assay does not indicate the directionality of motor movement. Previous studies have demonstrated that the velocities of filament-forming conventional myosins-II from either muscle or nonmuscle cells are comparable in both motility assays (Umemoto and Sellers: J. Biol. Chem. 265:14864-14869, 1990). However, similar studies using unconventional myosins are lacking. In the present report we have compared the rates of two structurally distinct unconventional myosins: brush border (BB) myosin-I and chick brain (CB) myosin-V (p190-calmodulin), using the sliding actin filament and Nitella-based in vitro motility assays. These two unconventional myosins differ from conventional myosins in that they appear unable to associate into bipolar filaments, and have extended rod-like neck domains which bind multiple calmodulin light chains in a Ca(2+)-sensitive manner.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Biology, Yale University, New Haven, CT 06511.
J S
JS
Wolenski
R E
RE
Cheney
P
P
Forscher
M S
MS
Mooseker
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D001921:Brain / Q000502:physiology; D002148:Calmodulin-Binding Proteins / Q000502:physiology*; D002645:Chickens; D000460:Chlorophyta; D066298:In Vitro Techniques; D008871:Microvilli / Q000502:physiology; D024701:Myosin Type V; D009218:Myosins / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000502:physiology*; D011506:Proteins / Q000502:physiology*
D002148:Calmodulin-Binding Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; C078829:chicken brain myosin-V p190; D024701:Myosin Type V; D009218:Myosins