J. Biol. Chem., Vol. 268, Issue 4, 2410-2415, 02, 1993
Enhanced stimulation of myosin subfragment 1 ATPase activity by addition of negatively charged residues to the yeast actin NH2 terminus
RK Cook, D Root, C Miller, E Reisler and PA Rubenstein
Department of Biochemistry, College of Medicine, University of Iowa, Iowa City 52242.
We examined the effects of yeast actin NH2-terminal mutations on actomyosin
interactions and the function of actin in vivo through measurements of
actin-activated ATPase activity, cosedimentation with rabbit muscle myosin
subfragment 1 (S-1), in vitro motility, and invertase secretion assays. As
reported earlier (Cook, R. K., Blake, W., and Rubenstein, P. A. (1992) J.
Biol. Chem. 267, 9430-9436), elimination of NH2-terminal acidic residues
from yeast actin results in an increased actin bundling, decreased
actin-activated S-1 ATPase, and complete inhibition of actin filament
sliding over myosin. Here we show that the addition of 2 new acidic
residues to the NH2 terminus of yeast actin increased the Vmax value and
the catalytic efficiency of the actin-activated ATPase activity of S-1.
However, the binding of actin to S-1 in the presence of ATP and the
velocities of actin sliding over myosin in the in vitro motility assays
were not affected by this mutation. Thus, the number of actin NH2-terminal
negative charges is important for actin activation of myosin S-1 ATPase
activity, while only a minimum number of acidic residues is required for
actin sliding over myosin in vitro. The number of actin NH2-terminal
negative charges therefore appears to determine the efficiency with which
the energy from ATP hydrolysis is converted to filament sliding.
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