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 det ermination 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 ind icate the directionality of motor movement. Previous studies have demo nstrated that the velocities of filament-forming conventional myosins- II from either muscle or nonmuscle cells are comparable in both motili ty 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 dis tinct unconventional myosins: brush border (BB) myosin-I and chick bra in (CB) myosin-V (p190-calmodulin), using the sliding actin filament a nd Nitella-based in vitro motility assays. These two unconventional my osins differ from conventional myosins in that they appear unable to a ssociate into bipolar filaments, and have extended rod-like neck domai ns which bind multiple calmodulin light chains in a Ca2+-sensitive man ner. Unlike conventional myosins, both of these unconventional myosins exhibit motility rates using the Nitella assay (BB myosin-I: approxim ately 10 nm/s; CB myosin-V: approximately 30 nm/s) that are 5- to 10-f old slower than that determined for these myosins using the sliding fi lament assay (BB myosin-I: 40-67 nm/s; CB myosin-V: 260-340 nm/s). (C) 1993 Wiley-Liss, Inc.