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Figure 2.
FIGURE 2. Biochemical characterization of S. pombe Arp2/3
complex with and without Arp2. A, effect of native and  Arp2
Arp2/3 complex on the time course of polymerization of
pyrene-labeled Mg-ATP actin. Conditions: 4 µM 15%
pyrene-labeled chicken skeletal muscle actin, 0.8 µM
SpWsp1-VCA, 200 µM complete SpArp2/3 complex ("complete")
or Arp2 Arp2/3 complex ("
Arp2") in 10 mM
imidazole, pH 7.0, 50 mM KCl, 1 mM MgCl[2], 1 mM EGTA, 0.13 mM
ATP, 63 µM CaCl[2], 0.3 mM DTT, 0.6 mM NaN[3] at 22
°C. Thick black line shows 4 µM actin and 0.8 µM
SpWsp1-VCA without Arp2/3 complex. Inset shows the concentration
of barbed ends when 50% of the actin was polymerized plotted as
a function of SpWsp1-VCA concentration for the complete SpArp2/3
complex (pool B). High concentrations of VCA decrease the rate
of polymer formation by inhibiting nucleation and slowing
pointed end elongation (23, 44). B, equilibrium binding of
rhodamine-labeled and unlabeled SpWsp1-VCA to Arp2
Arp2/3 complex and complete Arp2/3 complex measured by
fluorescence anisotropy. Conditions: 50 mM KCl, 10 mM imidazole,
pH 7.0, 1 mM MgCl[2], 1 mM EGTA, 0.1 mM ATP, 1 mM DTT, and 0.2%
thesit. Inset: titration of 100 nM SpWsp1-Rho-VCA with Arp2
(dashed line) and native Arp2/3 complex (solid line). The K[d]
values of SpWsp1-Rho-VCA were 120 ± 13 nM for the Arp2 and
49 ± 5 nM for complete Arp2/3 complex. Main plot:
titration of 100 nM SpWsp1-Rho-VCA and 300 nM Arp2
(dashed line) or native Arp2/3 complex (solid line) with
unlabeled SpWsp1-VCA. Curves were fit as described in the
methods giving K[d] values of 0.4 ± 0.1 µM and 0.9
± 0.1 µM for unlabeled SpWsp1-VCA binding the Arp2 and
complete complexes, respectively. C, fluorescence resonance
energy transfer to measure binding of SpWsp1-Rho-VCA to
OG-actin. Emission scans showing the dependence of the quenching
of the fluorescence of 100 nM OG-actin on the concentration of
SpWsp1-Rho-VCA in the same buffer as in B. Numbers below the
curves indicate Rho-VCA concentrations, in nanomolar. Samples
were excited at 480 nm. D, effect of Arp2 Arp2/3 complex and
native Arp2/3 complex on binding of OG-actin to Rho-VCA measured
by FRET as in C. Plots of fraction of OG-actin emission at 517
nm quenched verses Rho-VCA concentration. Fits of the data gave
a K[d] of 15.5 ± 1.7 nM for Rho-VCA binding to OG-actin
(solid line, open squares). In the presence of 3 µM native
Arp2/3 complex (solid line, filled circles), the K[d] increased
to 23.9 ± 1.3 nM. In the presence of 3 µM Arp2-less
complex (dashed line, filled triangles) the K[d] was 12.1
± 1.6 nM.
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