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Actin-based motor properties of native myosin VIIa

Departments of Pharmacology and Neurosciences, UCSD School of Medicine, La Jolla, California 92093-0983, USA

View larger version (42K): [in a new window]   Fig. 1. Calmodulin binding to myosin VIIa. (A) Diagram of the primary structure of native myosin VIIa, indicating the motor domain (head), the five IQ motifs (and potentially bound light chains (grey ellipses)), the coiled-coil domain (CC) and the tail, which includes the Myth4 and FERM domains (Chen et al., 1996; Weil et al., 1996). (B) Sequences of the five IQ motifs of human myosin VIIa (Hasson et al., 1995) compared with the IQ consensus motif. The number of asterisks indicates the extent of similarity of each motif to the consensus motif. Mouse myosin VIIa differs from the human at the following amino acids: N to H in IQ2; first Q to E and T to A in IQ4 (Mburu et al., 1997). (C) Immunoprecipitation of myosin VIIa and calmodulin from bovine retina with myosin VIIa antibodies. Preimmune serum (control Ab) and the N-terminal myosin VIIa antibody fail to immunoprecipitate myosin VIIa or calmodulin (lanes 1 and 2), whereas the tail myosin VIIa antibody immunoprecipitates both. (D) Release of calmodulin from immunoprecipitate. The beads containing the immunprecipitate (lane 1) were washed with buffer containing 1 mM EGTA (lane 2) or 10 µM Ca2+ (lane 3). More calmodulin is released in the presence of Ca2+, and this calmodulin characteristically has a slightly higher mobility in SDS-PAGE.

View larger version (58K): [in a new window]   Fig. 2. Immunoprecipitation of myosin VIIa from shaker1 mouse tissue. Western blot showing the result of immunoprecipitation of myosin VIIa from mouse testes of shaker1-null mutants (–/–) and control littermate heterozygotes (+/–).

View larger version (15K): [in a new window]   Fig. 3. ATPase activity of myosin VIIa. (A) A histogram illustrating the dependence of Mg2+ (2 mM) and actin filaments (2.5 µM) on myosin VIIa ATPase activity. (B) Relationship between myosin VIIa ATPase activity and the concentration of actin filaments. From these data a maximal ATPase activity (Kcat) of 4.3 s–1, with 7.0 µM of f-actin required for half-maximal activity (Kactin), was calculated.

View larger version (59K): [in a new window]   Fig. 4. In vitro motility of myosin VIIa. (A) Series of images illustrating movement of actin filaments by rabbit skeletal muscle myosin II (upper) and by mouse retinal myosin VIIa (lower). The white arrow indicates the starting point. The black arrow indicates the trailing end of a single actin filament. (B) Plot of the measured velocities of 50 individual actin filaments moved by mouse retinal myosin VIIa.

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