From the
The time course of interaction of caldesmon with actin may be
monitored by fluorescence changes that occur upon the binding of
12-( N-methyl- N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))-labeled
caldesmon to actin or to acrylodan actin. The concentration dependence
of the observed rate of caldesmon-actin binding was analyzed to a first
approximation as a single-step reaction using a Monte Carlo simulation.
The derived association and dissociation rates were 10
The protein caldesmon inhibits the stimulation of myosin ATPase
activity by actin (Dabrowska et al., 1985; Smith and Marston,
1985; Ngai and Walsh, 1985; Sobue et al., 1985). Caldesmon
also inhibits force production when added to smooth muscle fibers
(Pfitzer et al., 1993) and to skeletal muscle fibers, which
normally lack caldesmon (Brenner et al., 1991). Several
studies indicate that caldesmon has the potential to modulate
contractility (Yamashiro et al., 1990; Walker et al.,
1989; Hegmann et al., 1991; Katsuyama et al., 1992)
although its primary role as a regulatory protein is not yet proven.
We have proposed that an important aspect of the inhibition of
ATPase activity by caldesmon is the inhibition of binding of myosin
subfragment 1 (S1)
Testing the different models of caldesmon
both by mathematical modeling and by experimentation requires
information regarding the rates of binding of caldesmon to actin in the
presence and absence of tropomyosin. We have used two combinations of
fluorescent probes to measure the concentration dependence of this
association reaction. The concentration dependence of the observed
association rates was analyzed by a simple Monte Carlo model assuming
that the binding is a single-step mechanism.
For modification with
IANBD, caldesmon was treated with 10 mM dithiothreitol for 30
min at 37 °C and then loaded onto a 1.5
To determine
the location of the fluorescent probe, NBD-labeled caldesmon was
digested with chymotrypsin (1:1000, w/w) for 5 min, and the products of
reaction were analyzed by high pressure liquid chromatography on an
SP-R cation exchange column (Waters) or by SDS-polyacrylamide gel
electrophoresis. Polypeptides that had been previously determined to
originate from the NH
Protein concentrations were determined by absorbance at 280
nm except for caldesmon, which was determined by the Lowry assay using
bovine serum albumin as a standard. We have shown earlier that the
Lowry assay gives an excellent estimate of the true caldesmon
concentration (Velaz et al., 1989). The molecular weights used
for calculation of protein concentrations were 120,000 (S1), 42,000
(actin), 68,000 (tropomyosin), 16,500 (calmodulin), 87,000 (caldesmon),
and 20,000 (the actin binding fragments of caldesmon).
The concentration of caldesmon used was at least 10 times the
concentration of binding sites, assuming one caldesmon binds to at
least seven actin monomers. Two precautions were used to avoid actin
depolymerization in cases where very low actin concentrations were
used. Actin was diluted from a 20 µM stock and used for
kinetic measurements within 5 min so that appreciable depolymerization
would not occur. The same results were obtained using a final actin
concentration of 0.5 µM or 2 µM.
Alternatively, the actin was treated with a 1.2-fold molar excess of
phalloidin added from a 10 mM stock in methanol. Identical
results were obtained in the presence and absence of phalloidin.
Cytochalasin D (1 mol/100 mol of actin monomer) was sometimes added to
the actin when high actin concentrations were used to facilitate
mixing. We detected no difference in the traces obtained in the
presence and absence of cytochalasin D.
Association rates were
measured by mixing IANBD-labeled caldesmon with actin or
acrylodan-labeled actin in either the presence or absence of
tropomyosin. Averages of up to 15 kinetic measurements were analyzed by
fitting equations for a monoexponential or biexponential process to the
data with the software provided in the Applied Photophysics package.
Caldesmon binds to an actin lattice where a single binding site
consists of several actin monomers. In such situations there is a
parking problem, that is units consisting of seven free actin monomers
are not always available, so binding of caldesmon may require
reorganization of existing bound caldesmon molecules. The result is
that second order rate constants of the association reaction cannot
simply be obtained from the slope of k
The simulation of the
kinetics of binding of caldesmon to actin was carried out by following
the transitions of a single actin filament among its various
``states'' as a function of time, starting from a bare actin
filament at time 0. An actin state is specified not only by the number
of bound caldesmon molecules but also by the distribution of the gaps
between two neighboring caldesmons. The transition between any two
actin states always involves either a binding or a dissociation
reaction. Analyses of the equilibrium binding of caldesmon to actin by
the equation of McGhee and von Hippel (1974) show that there is a small
amount of positive cooperativity, which compensates for the parking
problem (Velaz et al., 1989). We neglect this cooperativity
between bound caldesmon molecules in this initial kinetic analysis.
Because the cooperativity is small, this is a good first approximation
(we are working on a more complete model that accounts for the
cooperativity). Thus, if the transition between two states involves the
binding of a caldesmon to a gap of m empty units between two
bound caldesmon molecules on actin, the transition probability can be
evaluated as follows:
At
any given state, all of the ``out-going'' transition
probabilities can be evaluated and used to calculate the average
``occupation time'' that the actin will stay in that state as
well as to determine which state the system will go to when the next
transition occurs. Thus, by following a path of state transitions, a
histogram of the total bound caldesmon on actin can be obtained as a
function of time (see Fig. 1). A large number of similar
histograms were obtained by repeating the simulation with the same set
of parameters. The kinetic curve of caldesmon binding was then obtained
by averaging over these histograms.
Light-scattering measurements were not useful for the
measurement of unmodified caldesmon binding to actin because the
initial binding reaction had a very small signal, while the subsequent
slow bundling of actin filaments (caused by the large excess of
caldesmon over actin) produced a very large signal. The slow reaction,
which was completed in about 20 min, was confirmed as actin filament
bundling by electron microscopy (not shown). Large signals were
obtained with the binding of NBD-labeled caldesmon to either
acrylodan-labeled actin or to native actin. In the former case, the
actin probe was observed, while in the latter case caldesmon
fluorescence was monitored.
Fig. 2A ( curves 1 and 3) shows that the binding of NBD-labeled
caldesmon quenched the fluorescence of acrylodan-labeled actin by 37%.
The binding of smooth muscle tropomyosin to acrylodan-labeled actin
resulted in an 18% increase in fluorescence ( curves 3 and 4). The fluorescence of the acrylodan-labeled
actin-tropomyosin complex was also greatly quenched by caldesmon-NBD
( curves 4 and 2). Fig. 2 B shows that S1 also quenched the fluorescence of acrylodan-labeled
actin. Thus while the acrylodan-modified actin gave a large signal upon
binding to caldesmon, its fluorescence was sensitive to other actin
binding proteins.
Since both the NH
The 35- and 20-kDa actin binding fragments of caldesmon, which
contain only one Cys residue, Cys
We had shown earlier that modification of caldesmon with
iodoacetamide does not alter its binding to actin or actin tropomyosin
(Velaz et al., 1989). Similarly, modification of the two Cys
groups of caldesmon with IANBD did not have a measurable effect on the
inhibitory activity of caldesmon (data not shown). Similarly,
modification of actin with acrylodan had little effect on the ATPase
activity (data not shown). For example, at 50 mM ionic
strength, smooth muscle tropomyosin enhanced the rate of ATPase
activity in the presence of acrylodan actin and absence of caldesmon by
a factor of 1.7; this is typical for unmodified actin (Williams et
al., 1984). Furthermore, more than twice as much caldesmon was
required to give 50% inhibition of the acrylodan actin-activated ATPase
activity in the absence of tropomyosin.
The rate of binding of
NBD-labeled caldesmon to either actin or acrylodan-labeled actin could
be readily observed in a stopped flow apparatus. Fig. 5shows an
example of a reaction using each probe. In the cases shown, the fit was
to a monoexponential function. The total fluorescence change was
completed within about 50 ms, and no further change occurred at longer
time intervals when actin bundles began to form. At higher
concentrations of caldesmon, a slightly better fit was obtained with a
biexponential fit; this complexity was ignored for this first analysis.
Although
fluorescence changes observed with caldesmon-NBD binding to pure actin
reflected changes in the caldesmon probe while binding to acrylodan
actin reflected changes in the actin probe, both measurements produced
similar kinetic profiles. Fig. 6 A shows the dependence
on the total caldesmon concentration of the observed half-time values
for a single exponential fit to the binding of caldesmon to pure actin.
Data obtained by observing the fluorescence of acrylodan actin
( squares) were indistinguishable from those obtained by
monitoring the caldesmon-NBD fluorescence ( circles). The
solid curve is the result of the simulation of these
data with a single-step model having an association rate constant,
k, of 1
Caldesmon-NBD is a useful model for studying the binding of
caldesmon to actin. This probe gave a large change in fluorescence upon
binding to actin but only small changes upon binding to myosin or
tropomyosin, thus indicating a high degree of selectivity. However,
observation of this probe on caldesmon had the disadvantage of high
background fluorescence when the caldesmon-NBD concentration was high
relative to actin. To overcome this difficulty, we have also monitored
acrylodan-labeled actin, which is quenched by the NBD probe on
caldesmon. Both probes left the steady state kinetics of ATP hydrolysis
unaltered, and similar transient kinetic results were obtained
regardless of whether the caldesmon probe or actin probe was monitored.
We have shown earlier that the binding of caldesmon to actin is
competitive with the binding of S1 in both the presence and absence of
tropomyosin (Chalovich et al., 1987; Hemric and Chalovich,
1988; Velaz et al., 1989, 1990; Chen and Chalovich, 1992). In
agreement with that result, we now report that the fluorescence change
due to caldesmon-NBD binding to actin was reversed by the binding of
substoichiometric amounts of S1 to actin both in the presence and
absence of tropomyosin. The region of caldesmon that binds to actin and
has inhibitory activity is at the COOH-terminal domain of caldesmon
(Szpacenko and Dabrowska, 1986; Fujii et al., 1987). This
region was also sufficient and necessary for the fluorescence changes
reported here.
The binding of caldesmon to actin is probably
mechanistically complex because of the parking problem that occurs for
binding of a large ligand to a lattice and because of the cooperativity
that may exist among adjacent ligands. The data in the present case
were analyzed by a Monte Carlo simulation that did not include
cooperative effects. The binding constants k and k`
are intrinsic rate constants for the binding to an isolated binding
site. Presumably, the true situation consists of a series of rate
constants depending on nearest neighbors and on the slower
rearrangement of sites to provide total saturation of the actin
filament. However, our simple analysis provided a reasonable
approximation to the actual event as judged by the close fit of the
theoretical curve to the data of Fig. 6. Such a simple analysis
would not be expected to be suitable for a more cooperative system such
as in the binding of tropomyosin to actin where
Tropomyosin is known to enhance the binding of
caldesmon to actin; this enhanced binding appeared, from the present
work, to be due to a decrease in the rate constant of dissociation of
caldesmon from actin. We did not observe any indication that
tropomyosin altered the mechanism of binding, that is the individual
time courses of the binding reaction were similar in the presence and
absence of tropomyosin; the concentration dependence of the half-times
also had a similar appearance. If there were two classes of binding
sites for caldesmon, in either the presence or absence of tropomyosin,
one might expect to see biexponential binding kinetics even at the
lowest caldesmon concentrations used since the caldesmon concentration
far exceeded the number of binding sites. In actuality, we observed a
good fit to the data with a single exponential curve except at high
caldesmon concentrations. While we cannot rule out multiple sites there
is no evidence from these data that they exist.
A more telling
experiment would be to measure the binding with excess actin over
caldesmon. However, this becomes experimentally difficult and
computationally nontrivial because several actin monomers constitute a
single binding site. Another approach is to measure the dissociation of
caldesmon from actin using the same probes described here. However,
analysis of this behavior requires Monte Carlo simulations that are
different from those presented here. This is a topic of current
investigation.
We thank Michael Vy-Freedman, Randal Hartwell, Hsiang
Chi Guo, and Dan Whitehead for expert technical assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
M
s
and 18.2
s
, respectively. Smooth muscle tropomyosin enhances
the binding of caldesmon to actin, and this was found to be due to a
reduction in the rate of dissociation to 6.3 s
.
There is no evidence from this study for a different mechanism of
binding in the presence of tropomyosin. The fluorescence changes that
occurred with the binding of 12-( N - methyl - N - (7
- nitrobenz-2-oxa-1,3-diazol-4-yl))-labeled caldesmon to actin or
actin-tropomyosin were reversed by the addition of myosin subfragment 1
as predicted by a competitive binding mechanism.
(
)
to actin in both the
presence and absence of tropomyosin (Chalovich et al., 1987;
Hemric and Chalovich, 1988; Velaz et al., 1989; Velaz et
al., 1990; Chen and Chalovich, 1992). This view is not universally
accepted, and other reports suggest that in the presence of tropomyosin
and under conditions where S1 concentrations are high relative to
caldesmon, inhibition of ATPase activity occurs with little reduction
of S1
ATP binding (Marston and Redwood, 1993). This latter model
requires that the mode of action of caldesmon be quite different in the
presence of tropomyosin.
Proteins
Smooth muscle tropomyosin was isolated
from turkey gizzards either by the method of Bretscher (1984) or by a
modification of the method of Graceffa (1992), which avoids heat
treatment of the tropomyosin. Other proteins were prepared as described
earlier (Chalovich et al., 1992).
15-cm column of ACA
202 (Spectrum) gel filtration resin equilibrated with 100 mM
KCl, 50 mM Tris-HCl, pH 7.5, 1 mM EDTA. To the
mixture was added a stock of 25 mM IANBD in dimethyl formamide
to give a concentration 5 times that of the caldesmon concentration.
The reaction was stopped by the addition of dithiothreitol to 1
mM after an incubation of 4 h at 18 °C. Unreacted probe
was removed by gel filtration chromatography on ACA 202 resin. The
extent of labeling was determined to be between 1.5 and 2 mol of
probe/mol of caldesmon using an extinction coefficient of 2.6
10
M
cm
at
495 nm (Trayer and Trayer, 1988). Actin was labeled with acrylodan at
Cys
by incubating at 4 °C for 12 h in a solution
containing 0.1 M NaCl, 10 mM Tris-HCl, pH 7.5, 2
mM MgCl
, and 1 mM NaN
with a
5-fold molar excess of acrylodan. The extent of labeling was
70-100% as determined using an extinction coefficient of 1.29
10
M
cm
at 360 nm (Prendergast et al., 1983).
-terminal myosin binding region (Velaz
et al., 1990) and the COOH-terminal actin binding region
(Chalovich et al., 1992) were fluorescently labeled. In
preparations that were not completely labeled, the myosin binding
fragment was more fluorescent than the actin binding fragments,
suggesting a more rapid reaction of the probe with the former site. In
another approach, the digested IANBD-labeled caldesmon was applied to a
1.5
9-cm calmodulin affinity column equilibrated with 5
mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM
MgCl
. The fluorescently labeled myosin binding fragment was
in the breakthrough volume and was purified further as described
earlier (Velaz et al., 1990). Intact caldesmon and the 35-kDa,
20-kDa, and smaller actin and calmodulin binding fragments were eluted
by replacing the buffer with one containing 1 mM EGTA in place
of the CaCl
. The 35- and 20-kDa actin binding fragments
were purified as described previously (Chalovich et al.,
1992).
ATPase Assays
The ATPase activity of skeletal S1
was measured by the liberation of P
from
[
P
]ATP as described earlier
(Chalovich and Eisenberg, 1982).
Sequence Analysis
Partially purified polypeptides
were run on SDS-10% polyacrylamide gels and electrophoretically
transferred to Bio-Rad sequential grade polyvinylidene difluoride
membrane. The fluorescent band was cut out of the membrane and
sequenced at the UCLA Protein Microsequencing Facility.
Electron Microscopy
Samples were prepared by the
drop method (Hayat, 1972) and negatively stained with 1% uranyl
acetate. Samples were examined with a JEOL 1200EX electron microscope
at 65 kV.
Steady State Fluorescence
Measurements
Fluorescence was measured on a SPEX Fluorolog 2
spectrofluorometer in 0.4 x 1.0-mm cells at 15 °C. The excitation
and emission slit widths were 1.25 mm (4.5-nm band-pass). Binding
isotherms were constructed by maintaining the actin concentration at 1
or 2 µM and varying the caldesmon concentration. Results
were most easily interpreted when the fluorescence of the actin probe
was monitored so that the signal was proportional to the number of
actin sites occupied. Curves of the fraction of caldesmon bound/total
actin, or , versus free caldesmon were constructed by
assuming that when the fluorescence no longer changed the actin was
saturated. The free caldesmon concentration was calculated using the
analysis of McGhee and von Hippel (1974) assuming different values of
stoichiometry ( n), isolated site binding constant
( K), and cooperativity parameter (
). These parameters
were varied until there was a satisfactory fit to the experimental
data. There is not a unique solution to such fluorescence titration
data when the stoichiometry is unknown. The stoichiometry of binding is
known to be 1 caldesmon per 7 (Velaz et al., 1989) or more
(Smith et al., 1987) actin monomers; a value of 7 was used in
the calculations. The MLAB program was used in this mathematical
modeling.
Stopped Flow Kinetic Studies
Measurements were
made in 10 mM imidazole, pH 7.0, 2 mM
MgCl, 34 mM potassium propionate, 1 mM
EGTA, and 1 mM dithiothreitol buffer, using an Applied
Photophysics DX17.MV/2 sequential stopped flow spectrofluorometer. The
binding of caldesmon to actin was monitored as an increase in light
scattering, an increase in fluorescence intensity of the NBD probe on
caldesmon, or a decrease in fluorescence of acrylodan-labeled actin
upon binding to IANBD-modified caldesmon. IANBD fluorescence was
monitored using a filter with 0% transmission at 510 nm and 80%
transmission at 540 nm with excitation at 492 nm. Acrylodan
fluorescence was monitored with excitation at 391 nm with a filter
having 0% transmission at 430 nm and 80% transmission at 500 nm or with
a 485-494-nm band-pass filter; both filters produced the same
rate constant, but the 500-nm cut-on filter produced the larger signal.
against
free ligand concentration. We therefore analyzed the kinetic data by a
Monte Carlo simulation. The binding and dissociation between a
caldesmon molecule and a 7-mer actin are assumed to be simple one-step
reactions with rate constants k and k`, respectively.
The equilibrium constant for the reaction is defined as K = k/ k`. Because the value of K can be obtained from equilibrium binding measurements, only one of
the rate constants is an adjustable parameter. We assumed that the
actins in solution are uniform in length, with M monomers per
actin filament. M and k (or k`) are the only
adjustable parameters in our simulation.
= ( m
6) kC
, where C
is the concentration of caldesmon in solution. Note that m must be larger than 7 for binding to occur. If the transition
involves the desorption of a caldesmon from the actin, the transition
probability is simply equal to k`. The number of caldesmon
molecules in solution changes when caldesmon binds to (or dissociates
from) actin; this causes the caldesmon concentration in solution to
change as a function of time during the simulation. The concentration
of caldesmon in solution at any given time can be evaluated using the
following equation: C
=
C
( n/ M)C
, where n is
the number of total bound caldesmon molecules on the single actin
filament and C
and C
are the initial concentrations of actin monomers and caldesmon
molecules, respectively, as prepared in the experiment at time 0.
Figure 1:
Example of a
typical histogram showing the total number of bound caldesmon molecules
on a single actin filament as a function of time during the first
(initial) few transitions of a Monte Carlo simulation. Each transition
involves the binding or dissociation of one caldesmon molecule only.
Therefore, the number of total bound caldesmon molecules increases or
decreases by one.
Figure 2:
Fluorescence emission spectra of
NBD-labeled caldesmon and acrylodan-labeled actin. Fluorescence was
measured with excitation at 391 nm in a solution containing 10
mM imidazole, pH 7.0, 2 mM MgCl, 34
mM potassium propionate, 1 mM EGTA, and 1 mM
dithiothreitol. A, effect of caldesmon-NBD and S1 on acrylodan
actin fluorescence. Curve 3 shows 1 µM
acrylodan actin alone, while curve 1 shows the effect
of the addition of 1.5 µM caldesmon-NBD. Curve 4 shows 1 µM acrylodan actin and 0.214
µM tropomyosin, while curve 2 shows the
effect of addition of 1.5 µM caldesmon-NBD. B,
effect of S1 on acrylodan actin fluorescence. Curve 2 shows 1 µM acrylodan actin, and curve 1 shows the effect of the addition of 2 µM
S1.
The fluorescence change that occurred when
caldesmon-NBD bound to acrylodan-labeled actin was related to the
fraction of actin monomers interacting with caldesmon-NBD.
Fig. 3
shows the binding curve derived from the decrease in
fluorescence of the actin-acrylodan as a function of the added
caldesmon-NBD. Analysis of the fluorescence data required the
assumption of a stoichiometry of binding. A reasonable fit to the data
could be obtained for any value of stoichiometry greater than 1:5; the
solid curve shown is for a stoichiometry of 1:7. Two
additional parameters describe binding of a large ligand to a lattice.
The binding constant for caldesmon binding to an isolated site of seven
actin monomers, K, was determined to be 3.2 10
M
. The parameter for cooperativity
between adjacent caldesmon molecules,
, was 17.9. The product
K
is the affinity of caldesmon to a site adjacent to an
existing bound caldesmon molecule; this was found to be 5.7
10
M
. In our detailed earlier
study, in which the binding was measured by a more direct method,
values of K and
were typically near 6
10
and 5-6, respectively, with K
equal to 3.6
10
M
(Velaz et
al., 1989). This agreement is reasonable and suggests that the
fluorescence change is monitoring the binding reaction.
Figure 3:
Fluorescence titration measurement of
binding of caldesmon-NBD to acrylodan actin. Fluorescence was measured
at 15 °C with excitation at 391 nm in the same buffer as in Fig. 2.
The fraction of actin containing bound caldesmon was assumed to vary in
a linear manner with the fluorescence change. The free caldesmon
concentration was calculated assuming a stoichiometry of binding of 1
caldesmon/7 actin monomers. The curve shown is the best fit of
the equation of McGhee and von Hippel (1974) to the data. The
parameters describing this curve are n = 7, K = 3.2 10
M
,
and
= 17.9 .
Fluorescence
changes accompanying the binding of IANBD-labeled caldesmon to pure
actin are shown in Fig. 4. In Fig. 4 A, curves 1 and 4 show that the binding of actin to
caldesmon-NBD resulted in a 71% increase in fluorescence intensity and
a small blue shift from 546 to 536 nm. The addition of a
substoichiometric amount of S1 to the actin-caldesmon complex resulted
in a partial reversal of the fluorescence enhancement ( curve 2); higher concentrations of S1 totally reversed the
fluorescence enhancement. This is consistent with our earlier
observation that caldesmon and S1 are competitive with each other for
binding to actin. The addition of ATP ( curve 3)
weakened the binding of S1 to actin and resulted in an increase in
fluorescence. This change was time-dependent, and the fluorescence
returned to a low value as the ATP was hydrolyzed and the S1 reattached
to actin (not shown).
Figure 4:
Fluorescence emission spectra of
NBD-labeled caldesmon. Fluorescence was measured with excitation at 492
nm with the same buffer as used in Fig. 2. A, effect of actin
and S1. Caldesmon-NBD (2 µM) is shown in curve 1. The fluorescence increases with the addition of 10
µM actin ( curve 4) and decreases again
with the further addition of 3.5 µM S1 ( curve 2). Dissociation of S1 with 1 mM ATP restores
the high fluorescence ( curve 3). B, effect
of S1 alone. Caldesmon-NBD (1.1 µM) is shown alone
( curve 1) and after the addition of 20
µM S1 ( curve 2). C, effect of
smooth muscle tropomyosin. Caldesmon-NBD (1.2 µM) is shown
alone ( curve 2) and after the addition of 3.1
µM tropomyosin ( curve 1). A mixture of
1.2 µM caldesmon-NBD and 10 µM actin is shown
in curve 3; the small fluorescence increase that
occurs with the addition of 3.1 µM tropomyosin is shown in
curve 4. D, the NBD-labeled 35-kDa,
COOH-terminal caldesmon fragment (1.2 µM) is shown in
curve 1. The addition of 4 µM actin
increases the fluorescence ( curve 3), while the
further addition of 3.4 µM S1 reverses this increase
( curve 2).
Caldesmon binds weakly to S1 (Hemric and
Chalovich, 1988). Fig. 4 B shows that a small decrease in
fluorescence occurred upon the addition of S1 to caldesmon-NBD in the
absence of actin. Caldesmon also binds to pure tropomyosin (Graceffa,
1987; Fujii et al., 1988; Watson et al., 1990).
Fig. 4C shows that tropomyosin caused a small
(8-9%) increase in fluorescence intensity in both the absence
( curves 1 and 2) and presence ( curves 3 and 4) of actin (Fig. 4 C). All
of these changes were much smaller than those that occurred when
caldesmon-NBD bound to actin.
and COOH
ends of caldesmon-NBD contained a fluorescent label it was necessary to
determine if the observed fluorescence change was due solely to the
interaction of the COOH-terminal actin binding region with actin. The
COOH- and NH
-terminal polypeptides of IANBD-labeled
caldesmon were isolated from chymotryptic digests of the caldesmon.
Amino-terminal sequence analysis of the presumptive myosin binding
fragment indicated the presence of two polypeptides,
Ser
-Tyr-Gln-Arg-Asn-Asp-Asp and
Gln
-Arg-Asn-Asp-Asp-Asp, that confirmed that this fragment
is from the NH
-terminal region containing
Cys
. The addition of actin did not result in a change in
the fluorescence of the labeled NH
-terminal fragment.
, were also purified and
found to be fluorescent. Fig. 4 D shows that addition of
actin to the pure IANBD-labeled 35-kDa caldesmon fragment resulted in a
92% increase in fluorescence ( curves 1 and
3). The increase in fluorescence was largely eliminated by the
addition of substoichiometric concentrations of S1 ( curve 2). Therefore, the change in fluorescence of caldesmon
was due to the interaction of the COOH-terminal region of caldesmon
with actin.
Figure 5:
Rates of fluorescence change for the
binding of caldesmon-NBD with actin and acrylodan actin. Fluorescence
was measured in the same buffer as in Fig. 1 and at 15 °C.
Curve A is for binding of 0.5 µM
caldesmon-NBD to 0.3 µM acrylodan actin (0.043
µM sites) and shows the decrease in acrylodan
fluorescence. The fitted curve is for a rate of 68
s. Curve B is for the binding of
0.45 µM caldesmon-NBD to 0.3 µM unmodified
actin (0.043 µM sites) and shows the increase in NBD
fluorescence. The fitted curve is for a rate of 49
s
.
The analysis of the rate data to obtain the rate constants k and k` was done by a Monte Carlo simulation using a
simple one-step binding model. For this analysis, we assumed binding
constants of caldesmon to actin in the absence and presence of
tropomyosin of 0.55 10
M
and 1.9
10
M
,
respectively (Chen and Chalovich, 1992). These binding constants are
those to an isolated caldesmon binding site composed of seven actin
monomers. The actin lengths assumed in the simulations were M = 700, 1400, 2800, and 5600. The half-times of the
individual reactions were found, in simulations, to be rather
insensitive to the actin length. Thus, most of our simulations were
based on M = 700 to save computing time.
10
M
s
and a dissociation rate constant,
k`, of 18.2 s
. While this was the best fit
of the data, most of the data fell within an envelope bracketed by
theoretical curves with association rate constants ranging from 0.8 to
1.2
10
M
s
. The simple model used was a good first
approximation of the binding.
Figure 6:
Caldesmon concentration dependence of the
half-time of binding of caldesmon-NBD to actin ( squares) and
acrylodan actin ( circles) in the absence ( A) and
presence ( B) of smooth muscle tropomyosin. Values of
from data such as that shown in Fig. 5 were plotted against the total
caldesmon concentration. Binding was measured at 15 °C in the same
buffer used in Fig. 2. A, in the absence of tropomyosin, the
simulated curve shown is for k = 1
10
M
s
and
k`= 18.2 s
. B, in the
presence of smooth muscle tropomyosin the simulated curve shown is for k = 1.2
10
M
s
and
k`= 6.3
s
.
Fig. 6B shows that the
addition of tropomyosin did not greatly alter the observed kinetics of
caldesmon binding. The results of experiments done by monitoring
acrylodan actin ( circles) and caldesmon-NBD ( squares)
were in close agreement with each other, indicating that the same
process was being monitored in each case. The profile of
versus caldesmon concentration was quite similar to that
obtained in the absence of smooth muscle tropomyosin. The best fit of
the simulated data was with an association rate constant of 1.2
10
M
s
and
dissociation rate constant of 6.3 s
, and the data
were bracketed by curves generated with association rate constants of 1
and 1.4
10
M
s
. Interestingly, the enhanced binding of
caldesmon to actin in the presence of tropomyosin appeared to be due
primarily to a decrease in the rate constant for caldesmon detachment;
the association rate constant was relatively unchanged. The values of
k and k` that generate the best fit of half-times
with experimental points are tabulated in .
= 1600
(Wegner, 1979). Even in the present case, the inclusion of
cooperativity would have given a better fit of the individual reactions
run at high caldesmon concentrations where there was a slight deviation
from a single exponential. We will include the effect of cooperativity
in the future.
Table: Rate constants for the binding of caldesmon
to actin in the presence and absence of tropomyosin
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.