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Figure 1.
(a) Effect of PBP on the steady-state actin-activated ATPase
activitiy of Dd myosin-5b. Actin-activated ATPase activities in
the absence (  square
) and presence of 25  M
PBP (  down
triangle ) are shown. (b) Concentration-dependence of the
inhibition of the actin-activated ATPase activity of different
myosin isoforms in the presence of 0–150 M
PBP. The semilogarithmic plot shows the concentration dependence
for myosin-1E (o), myosin-2 ( square
), Dd myosin-5b (  )
and chicken myosin-5a ( down
triangle ). The concentrations of PBP required for half-maximal
inhibition (IC[50]) of the different myosin motors were
determined from sigmoidal fits of the data. All parameters are
summarized in Table 1. (c) Fluorescence transients obtained upon
mixing 1 M
Dd myosin-5b with 10 M
mantATP in the absence and presence of 5 M,
25 M,
50 M
and 100 M
PBP. The concentration dependence of the amplitude of the fast
phase is shown in the inset. (d) Rate constants for ATP binding
and hydrolysis were determined by following the time-dependent
changes in the intrinsic protein fluorescence of Dd myosin-5b.
Addition of 25 M
PBP leads to a 14-fold reduction in the apparent second-order
rate constant for ATP binding and a ten-fold reduction of the
rate of ATP hydrolysis (Supplementary Methods online). (e)
PBP-mediated inhibition of chicken myosin-5a in the in vitro
motility assay. The histograms show the average sliding velocity
of rhodamine-phalloidin–labeled actin filaments in the absence
and presence of 10 M
PBP.
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