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INTRODUCTION |
Smooth muscle myosin
(SMM),1 like other members of
the myosin II family, has two heads connected by a coiled-coil tail.
SMM and the double-headed subfragment HMM are regulated by
phosphorylation of the two regulatory light chains, one on each head
(1-3). In contrast, single-headed SMM (4, 5) and the single-headed S1
(6-8) are not regulated by phosphorylation. Non-muscle HMM IIB is also
regulated by phosphorylation, and constructs lacking one motor domain
have been shown to be unregulated (9). Therefore, two motor domains are
required for regulation.
Structural differences between unphosphorylated and phosphorylated HMM
have been demonstrated by a number of studies. Reconstruction of images
of expressed unphosphorylated HMM in the presence of ATP in
two-dimensional crystalline arrays (10, 11) shows an asymmetrical
structure with the converter domain of one head bound to the
actin-binding site of the other head. No interaction was seen between
the motor domains of phosphorylated HMM. This model is supported by
data from Berger et al. (12), who demonstrated that only one
of the heads of an unphosphorylated expressed smooth muscle HMM-ADP
complex binds to actin. Either the binding of the first head
prevented the binding of the second head or the second head bound
weakly such that no signal was observed for its binding. Both heads
bound to actin in the phosphorylated state. These data are inconsistent
with two studies of the effect of ADP and phosphorylation in intact
gizzard muscle, which are consistent with binding of both heads of
myosin to actin irrespective of ADP or phosphorylation (13, 14).
We investigated the actin binding properties of HMM derived from
chicken gizzards. Digestion of SMM by Staphylococcus aureus V8 protease or chymotrypsin generates HMM with varying degrees of
internal cleavage at loop 2 (the actin-binding loop), but the cleavage
products remain associated under non-denaturing conditions (6, 15-19).
Based upon the model discussed previously, it was possible that the
extent of internal heavy chain cleavage at the actin-binding loop could
alter the actin binding behavior. Therefore, we produced HMM with
between 5 and 95% heavy chain cleavage for this study. Measurements of
the fluorescence changes upon binding of HMM to pyrene-actin and upon
ATP-induced dissociation from pyrene-actin were used to determine the
stoichiometry of HMM binding to actin in the unphosphorylated and
thiophosphorylated states. We show that both heads of tissue-derived
HMM bind to actin. This two-headed binding was observed irrespective of
the extent of internal heavy chain cleavage, the presence or absence of
ADP, or the phosphorylation state of the RLC. These experiments were consistent with the fact that ADP did not induce dissociation of
the pyrene-actin HMM complex irrespective of the phosphorylation state.
As these data contrast those for an expressed HMM construct (12),
explanations for the differences between the two protein preparations
are presented.
We also measured the actin-activated ATPase activity of
unphosphorylated and thiophosphorylated HMM by single turnover assays with both ATP and FTP. All HMM preparations used in this study were
found to be fully regulated as defined by a slow turnover rate in the
presence of actin for the unphosphorylated protein. Therefore, we have
shown that the native tissue-derived unphosphorylated HMM-ADP complex
binds to actin with two heads. The one-headed actin binding mode of an
unphosphorylated HMM-ADP complex predicted by the model of Wendt
et al. (10, 11) is not a property required for
down-regulation.
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EXPERIMENTAL PROCEDURES |
Protein Preparations--
SMM was prepared from frozen chicken
gizzards (20) obtained from Pel-Freez Biologicals (Rogers, AR). HMM and
S1 with between 50 and 60% cleaved heavy chain was obtained by
S. aureus protease V8 digestion of SMM in the presence
of ATP as described previously (6, 21). HMM with 95% cleaved heavy
chain was prepared by digestion in the absence of ATP. HMM with 25% or
less than 5% heavy chain cleavage was prepared by limited digestion
with chymotrypsin (Sigma) as described (18) except that digestion was
performed with 0.05 mg/ml chymotrypsin at 25 °C for 3 min.
Chymotryptic digestion also results in cleavage of the RLC. Therefore,
cleaved RLC was exchanged for intact, wild-type RLC using a standard
exchange procedure followed by purification of fully exchanged HMM
(with two intact RLC) as described previously (21). Myosin, HMM, and S1
concentrations were determined using the following 0.1% extinction coefficients: 0.56, 0.65, and 0.75, respectively. The extent of heavy
chain cleavage of HMM and S1 preparations was estimated by
densitometric scanning and analysis of SDS gels (Novex precast 4-20%
acrylamide gradient Tris-glycine (Invitrogen)) using an EPI Chem II
darkroom and Labworks image analysis software (UVP Laboratory
Products, San Gabriel, CA). All HMM preparations were stored on ice and
used within 2 weeks of purification. F-actin was prepared from rabbit
muscle according to the method of Spudich and Watt (22) and was labeled
with pyrene iodoacetamide (Molecular Probes) (23). MLCK was prepared
from frozen chicken gizzards by the method of Adelstein and Klee (24)
except that Superdex 200 (Amersham Biosciences) was used for gel
filtration, and Super Q (Toso-Hass) was used for anion exchange.
N-terminal Sequencing--
Protein bands were transferred
from an SDS gel to a PVDF membrane, and the sequences of the largest
HMM fragment (see Fig. 1, heavy chain) and the N-terminal
fragment (see Fig. 1, N) were determined using an Applied
Biosystems Procise-HT 492. The transfer efficiency was >80%.
Thiophosphorylation of Proteins--
HMM was thiophosphorylated
by incubation in 50 mM NaCl, 2.5 mM
MgCl2, 2.5 mM CaCl2, 4 µg/ml
calmodulin (Sigma), 1 mM ATP
S (Roche Molecular
Biochemicals), and 20-40 µM MLCK. Excess ATP
S was
removed by Sephadex G-50 (Sigma) centrifugal gel filtration (25). The
extent of thiophosphorylation was >95% as verified by gel
electrophoresis as described previously (26) except that protein
samples were applied to Novex precast 10% acrylamide Tris-glycine gels (Invitrogen).
Transient Kinetics--
Experiments were carried out using a
Hi-Tech SF-61 DX2 stopped-flow spectrophotometer equipped with a
150-watt mercury-xenon lamp and an electronic shutter to avoid
photolysis over prolonged time courses. All HMM and S1 concentrations
refer to the concentration of heads. All pyrene-actin solutions
contained equimolar phalloidin (Sigma) to stabilize the F-actin form.
Pyrene-actin fluorescence was excited at 365 nm, and emission was
detected after passing through a KV-399 cut-off filter (Schott).
ATP-induced dissociation experiments were performed using the method of
Kurzawa and Geeves (27). All concentrations quoted refer to
concentrations in the syringes before mixing at a 1:1 ratio. Mixtures
of pyrene-actin and HMM for dissociation experiments were preincubated
for 30 min at 20 °C to ensure that the mixture had reached
equilibrium. For association experiments, all concentrations quoted
refer to the concentration in the cuvette after mixing. Data were
analyzed by fitting the average from three to four shots (with a
variance of between ±0.3% and ±5%) to a single or double exponential model using the KinetAsyst 2 curve fitting software (Hitech
Scientific, Salisbury, UK). To allow comparison between different data
sets, fluorescence changes for dissociation experiments were expressed
as the ratio of the fluorescence amplitude change to the final
pyrene-actin fluorescence
(
F/Ffinal). Fluorescence changes
for association experiments were expressed as the ratio of the
fluorescence amplitude change to the initial pyrene-actin fluorescence
(
F/Finitial). Since all plots of
F/Ffinal and
F/Finitial versus HMM
concentration showed a clear plateau, the
Fmax/Ffinal and
Fmax/Finitial were
estimated from the highest HMM concentration tested. Association and
dissociation experiments were carried out at 20 °C in 20 mM MOPS (pH 7.0), 5 mM MgCl2, 100 mM KCl, 0.1 mM EGTA, and 1 mM
DTT.
All single turnovers were performed at 25 °C in 10 mM
MOPS (pH 7.0), 50 mM KCl, 0.1 mM EGTA, 1 mM DTT. FTP single turnovers were performed as described
previously (9), and ATP single turnovers were performed as follows. In
stopped-flow double-mixing experiments, syringe 1 contained 1.6 µM unphosphorylated HMM heads (60% of heavy chain
cleaved), and syringe 2 contained 1.6 µM ATP. The
contents of syringe 1 and syringe 2 were mixed in a 1:1 ratio and
incubated for 10 s. This aged mixture was then mixed with 10 µM actin containing 80 µM mant-ATP. Final
conditions in the cuvette were therefore 0.4 µM HMM
heads, 0.4 µM ATP, 5 µM actin, and 40 µM mant-ATP. Upon this second mix, ATP is hydrolyzed, and Pi and ADP are released, thus allowing mant-ATP to bind.
Fluorescence of the mant-ATP (measured through a KV 399 filter) at the
active site is enhanced by energy transfer from the excited active site tryptophan (excited at 295 nm). The increase in fluorescence upon mant-ATP binding was used to monitor the rate of ATP turnover.
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RESULTS |
Preparation of HMM with Various Degrees of Heavy Chain
Cleavage--
HMM prepared proteolytically from SMM contains heavy
chains cleaved internally at the actin-binding loop. However, cloned, expressed HMM contains only intact, full-length heavy chains. To
examine what effect, if any, heavy chain cleavage at the actin-binding loop had upon the mode of actin binding, we prepared tissue-derived HMM
with between 95 and 5% heavy chain cleavage (Fig.
1). Lanes 1-3 show three
preparations of HMM with 95, 60, or 50% of the heavy chain cleaved by
V8 protease. Chymotryptic digestion was required to achieve
preparations with 25 and 5% heavy chain cleavage (Fig. 1, lanes
4 and 5).

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Fig. 1.
SDS-PAGE of HMM
preparations with various extents of heavy chain cleavage.
Gel 1 (lanes 1-3): lane 1, HMM (95%
cleaved) prepared with V8 protease in the absence of ATP; lanes
2 and 3, HMM (60 and 50% cleaved, respectively)
prepared with V8 protease in the presence of ATP. Gel 2 (lanes 4 and 5): lanes 4 and
5, HMM (25 and 5% cleaved, respectively) prepared with
chymotrypsin followed by RLC exchange. Uncleaved heavy chain
(HC), N-terminal (N) and C-terminal
(C) fragments generated by cleavage, RLC, and essential
light chain (ELC) are indicated.
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The largest fragment (Fig. 1, HC) and the N-terminal
fragment (Fig. 1, N) were transferred to a membrane and
sequenced. These data showed that residues 1-9 and 1-27 for V8 and
chymotryptic HMM, respectively, were cleaved from the N terminus. It
was difficult to determine the extent of this cleavage since a
gel shift was not observed (Fig. 1). For 5% cleaved chymotryptic HMM
(lane 5, HC), we measured 2 pmol of the sequence
beginning at residue 28. If all of the heavy chain were N-terminally
cleaved, we would have measured ~50 pmol. Therefore, we estimate that
only 5-10% of the N terminus is cleaved in this preparation.
ATP-induced Dissociation of the acto-HMM Complex--
Binding of
smooth muscle S1 to pyrene-labeled actin quenches the pyrene
fluorescence by ~75% (28), as has been found for skeletal muscle S1
(23, 27). Addition of ATP to the acto-HMM complex dissociates the heads
from pyrene-actin, resulting in an increase in pyrene fluorescence; the
amplitude of the fluorescence change is dependent upon the amount of
pyrene-actin-HMM complex present before the addition of ATP. Fig.
2 shows representative traces of
ATP-induced dissociation of pyrene-actin complexed with u-HMM or
u-HMM-ADP showing an ~75% change in fluorescence. The u-HMM-ADP
complex dissociated more slowly (Fig. 2) because ADP must dissociate
before ATP can bind, but ADP had little effect upon the
F.

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Fig. 2.
ATP induced dissociation of u-HMM from
pyrene-actin in the presence or absence of ADP. Representative
traces are shown for the fluorescence changes upon mixing 80 nM pyrene-actin and 80 nM u-HMM heads (with
50% cleaved heavy chain), in the presence and absence of 200 µM ADP, with 80 µM ATP. All concentrations
are before mixing.
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Fig. 3 shows the HMM concentration
dependence of the fluorescence increase upon ATP-induced dissociation
of u-HMM (with 5% cleaved heavy chain) from pyrene-actin. The
amplitude of the fluorescence change was linearly dependent upon the
HMM concentration until a plateau was reached at ~80 nM
HMM heads in the presence of 80 nM actin. The maximal
fluorescence change
(
Fmax/Ffinal) was 0.80 (Fig. 3 and Table I). S1, which has no
partner head to constrain binding to actin, was used as a
control for the fluorescence changes to be expected upon complete
saturation of pyrene-actin with heads. The expected
Fmax/Ffinal was
observed (Table I, 0.74). Therefore, these data for HMM (Fig. 3) are
consistent with stoichiometric binding of both heads to pyrene-actin
with a dissociation constant (Kd)
80 nM. This means that both heads of a single HMM molecule are
binding to actin and that all of the actin-binding sites eventually
become filled with heads. Fig. 3 shows that the u-HMM-ADP complex
behaves in a similar manner. The HMM concentration dependence of
fluorescence changes for u-HMM, tp-HMM, and S1 (with 50% cleaved heavy
chain) were also measured, in the presence and absence of ADP (plots
not shown), and these data are tabulated in Table I. For all proteins
studied, a plateau in the
F/Ffinal was reached at HMM concentrations less than 1.5× the actin
concentration. These data are consistent with stoichiometric binding of
both heads to pyrene-actin (Table I). The
kobs values of u-HMM and tp-HMM dissociation
from pyrene-actin were monophasic and were independent of the HMM
concentration. The kobs = 29 s
1
and 28 s
1 in the absence of ADP, and the
kobs = 2.2 s
1 and 1.6 s
1 in the presence of ADP, for u-HMM and tp-HMM,
respectively.

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Fig. 3.
Fluorescence amplitudes for ATP-induced
dissociation of u-HMM from pyrene-actin versus u-HMM
concentration. Pyrene-actin (80 nM) and the indicated
concentrations of 5% cleaved chymotryptic u-HMM (squares)
or the u-HMM-ADP complex (circles; formed by addition of 200 µM ADP) were mixed with 80 µM ATP. All
concentrations are before mixing. The amplitudes of the pyrene-actin
fluorescence changes ( F/Ffinal)
are plotted against the concentration of HMM. The intersecting lines
were drawn by eye through the data for HMM (squares). The
buffer conditions are 100 mM KCl, 20 mM MOPS
(pH 7.0), 5 mM MgCl2, 0.1 mM EGTA,
1 mM DTT.
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Table I
Maximal fluorescence changes upon mixing nucleotide (ATP or ADP) with
the complex of pyrene-actin and S1, u-HMM, or tp-HMM
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Effect of ADP upon the acto-HMM Complex--
There was no change
in fluorescence upon mixing the pyrene-actin-HMM complex (either tp-HMM
or u-HMM) with saturating concentrations of ADP (Table I). This result
means that the extent of actin binding was not significantly affected
by ADP under these conditions. These data are consistent with the
ATP-induced dissociation results (Fig. 3) and suggest that both heads
of HMM bind to actin in the presence of ADP.
Association of HMM with Pyrene-Actin--
Fig.
4 shows representative traces of u-HMM or
u-HMM-ADP (60% cleaved heavy chains) association with pyrene-actin.
Fig. 5 shows the fluorescence decreases versus HMM
concentration for u-HMM or
u-HMM-ADP (with 5% heavy chains cleaved). The amplitudes of
the fluorescence changes were linearly dependent upon the HMM concentration until a plateau was reached at ~0.5 µM
heads in the presence of 0.5 µM actin. The maximal
fluorescence changes (Table II,
Fmax/Finitial) are
consistent with stoichiometric binding of both heads to pyrene-actin,
in the presence or absence of ADP. Therefore, we observed the same
stoichiometry that was observed for the dissociation experiment (Fig.
3) as expected. We did the same experiment for u-HMM
preparations with 50% heavy chain cleavage at three different KCl
concentrations, for u-HMM with between 25 and 95% heavy chain
cleavage, and for tp-HMM and S1 with 60% heavy chain cleavage (plots
not shown). For all proteins and conditions studied, a plateau in the
F/Finitial was reached at a
Fmax/Finitial of
0.62-0.90 as tabulated in Table II, at HMM concentrations less than
1.5× the actin concentration, consistent with binding of both heads of
HMM to pyrene-actin. As the extent of heavy chain cleavage increased,
the HMM concentration at which a plateau was reached also increased
(plots not shown), consistent with weakening of the actin binding
affinity as shown previously for proteolytic smooth muscle S1 by
Ikebe et al. (19).

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Fig. 4.
Association of u-HMM with pyrene-actin in the
presence and absence of ADP. Representative traces are shown for
the fluorescence changes upon mixing 0.5 µM u-HMM (with
60% cleaved heavy chain), in the presence and absence of 100 µM ADP, with 0.5 µM pyrene-actin. All
concentrations are after mixing.
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Fig. 5.
Fluorescence amplitudes for association of
u-HMM with pyrene-actin versus HMM concentration. Pyrene-actin
(0.5 µM) and the indicated concentrations of 5% cleaved
chymotryptic u-HMM (squares) or the u-HMM-ADP complex
(circles; formed by addition of 100 µM ADP)
were mixed. All concentrations are after mixing. The amplitudes of the
pyrene-actin fluorescence changes
( F/Finitial) are plotted against
the concentration of HMM. The intersecting lines were drawn by eye
through the data for u-HMM (squares). The buffer conditions
are 100 mM KCl, 20 mM MOPS (pH 7.0), 5 mM MgCl2, 0.1 mM EGTA, 1 mM DTT.
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Table II
Maximal fluorescence changes upon association of S1, u-HMM, and tp-HMM
with pyrene-actin
Data were obtained from an experiment similar to that shown in Fig. 5.
Fmax/Finitial was determined
from the average fluorescence change
( F/Finitial) at 1.5 µM
HMM heads. The buffer conditions were 20 mM MOPS (pH 7.0),
5 mM MgCl2, 0.1 mM EGTA, 1 mM DTT, with NaCl concentrations as indicated. V8 is
S. aureus protease.
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In the absence of ADP, association of u-HMM and tp-HMM was monophasic
at the lowest HMM concentrations tested in Fig. 5 (data not shown).
This was expected if the association process for each head was
equivalent under conditions in which actin is not limiting. A biphasic
process was observed at the higher u-HMM or tp-HMM concentrations as
expected under conditions of limiting actin availability. The
tp-HMM-ADP complex behaved as described above for tp-HMM. In contrast,
the u-HMM-ADP complex showed biphasic actin association kinetics with
similar amplitudes for each rate, even at the lowest HMM concentrations
in which actin was not limiting.
Single Turnover of MgFTP in the Presence of Actin--
A number of
recent studies have demonstrated that steady-state ATPase assays are an
unsatisfactory method for determining the true degree of regulation of
smooth and nonmuscle myosins (21, 29, 30). We therefore assessed
regulation by using a single turnover stopped-flow assay with the
fluorescent ATP analog, FTP, for all HMM preparations used in this
study (data not shown). The rate of FTP single turnover for all
preparations was monophasic at ~0.001 s
1 at 5 µM actin, consistent with our previous measurements (21). In addition, we measured the rate of FTP single turnover
versus actin concentration for u-HMM and tp-HMM with 40%
heavy chain cleavage (Fig. 6). Data for
tp-HMM fit best to a double exponential model, whereas data for u-HMM
fit best to a single exponential model. The fast rate of tp-HMM was fit
to the Michaelis-Menten equation, giving a Vmax
of 2.9 ± 0.2 s
1 and a
Katpase of 40 ± 8 µM. The
rates for u-HMM were low over a wide range of actin concentrations and
could not be fit to the Michaelis-Menten equation. At 90 µM actin, the rate for u-HMM was 0.005 s
1,
whereas the rate for tp-HMM was 2 s
1, representing a
400-fold increase in activity upon thiophosphorylation. In a previous
study, we measured the steady-state MgATPase activities of tp-HMM (21).
The Vmax and Katpase of
tp-HMM were 2.3 s
1 and 31 µM, respectively,
consistent with the single turnover values presented here. Our results
(Fig. 6) are similar to single turnover data obtained for an
expressed thiophosphorylated non-muscle HMM IIB construct that has no
heavy chain cleavage (9).

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Fig. 6.
FTP single turnover rates for u-HMM and
tp-HMM versus actin concentration. Rates of phosphate release from
the acto-HMM-FDP-Pi complex were measured for an HMM
preparation with 40% cleaved heavy chain. Final conditions for the
turnovers were: 0.4 µM HMM heads, 0.4 µM
FTP, 100 µM ATP, 0.4 mM MgCl2,
and various actin concentrations in 10 mM MOPS (pH 7.0), 50 mM NaCl, 0.1 mM EGTA, 1 mM DTT at
25 °C. Data for u-HMM (triangles) were fit to a single
exponential. Data for tp-HMM were fit to a double exponential
(circles, fast rate; squares, slow rate). A
Michaelis-Menten fit to the fast rate (straight line) gave
Vmax = 2.9 ± 0.2 s 1 and
Katpase = 40 ± 8 µM.
Katpase is the actin concentration at
half-maximal ATPase activity.
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As an alternative method to assess regulation, the turnover rate of the
physiological nucleotide, ATP, was measured for u-HMM with 60% cleaved
heavy chain (Fig. 7). Data were fit to a
single exponential plus a line and gave a rate of 0.0027 s
1 at 5 µM actin. The linear process had a
slope of 0.0008. A faster phase was not detected. With FTP as the
nucleotide substrate (Fig. 6), the equivalent rate at 5 µM actin for u-HMM was 0.001 s
1. Therefore,
the FTP and ATP turnover rates were within a factor of 3 and were
consistent with full regulation of turnover with both ATP and FTP.

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Fig. 7.
Single turnover of ATP by u-HMM. A
representative trace is shown for the turnover of ATP. Final
concentrations were 0.4 µM u-HMM heads (60% cleaved),
0.4 µM ATP, 5 µM actin, and 40 µM mant-ATP in 10 mM MOPS (pH 7.0), 50 mM KCl, 0.1 mM EGTA, 1 mM DTT at
25 °C.
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DISCUSSION |
We have demonstrated that both heads of tissue-derived HMM and
HMM-ADP bind to actin, irrespective of the phosphorylation state of the
RLC. These findings are consistent with the effects of ADP and
phosphorylation upon measurements of RLC mobility (13) and tension (14)
in intact smooth muscle. In contrast, Berger et al. (12)
found that only one head of expressed smooth muscle u-HMM-ADP bound to
actin, whereas tp-HMM, tp-HMM-ADP, and u-HMM bound with two heads. The
buffer conditions and protein concentrations used in this study and the
Berger et al. (12) study were similar. Berger et
al. (12) used the same ATP-induced dissociation method that we
used here. Therefore, it appears that the HMM preparations used in the
two studies are different.
One obvious difference between the HMM preparations is the presence of
internal heavy chain cleavage at the actin-binding loop in
tissue-derived HMM. In our HMM preparation with 5% heavy chain
cleavage, a maximum of 10% of all molecules would contain at least one
cleaved head with at least 90% of all molecules containing two
uncleaved heads. If these uncleaved u-HMM-ADP molecules behaved in a
manner similar to the expressed u-HMM-ADP, the
Fmax/Ffinal (Table I)
or
Fmax/Finitial
(Table II) for u-HMM-ADP would be 45% lower than that for u-HMM. Our
data for u-HMM with 5% heavy chain cleavage show no significant
differences between maximal fluorescence amplitude changes
obtained in the presence or absence of ADP using two different methods
(Fig. 3 and Table I, Fig. 5 and Table II). We conclude that the extent
of heavy chain cleavage at the actin-binding loop is not the
reason for the differences between expressed and tissue-derived
HMM.
In addition to the actin-binding loop cleavage, chymotrypsin and V8
protease cleave a small number of residues from the N terminus of the
heavy chain. The V8 protease preparation was missing only 9 residues
and is unlikely to explain the different actin binding behavior. For
5% cleaved chymotryptic HMM, 27 residues are cleaved, but we estimated
that only 5-10% of the N terminus is cleaved. This suggests that
N-terminal cleavage is not likely to explain the differences in actin
binding behavior by the same reasoning described above for the
actin-binding loop.
Our tissue-derived HMM was not frozen at any stage of the preparation,
whereas the expressed HMM was frozen in liquid nitrogen in the presence
of sucrose and stored at
80 °C. We have found that freezing
tissue-derived HMM in this manner causes loss of regulation. However,
both Berger et al. (12) and this study (Figs. 6 and 7)
showed that HMM preparations were regulated using single turnover approaches.
Berger et al. (12) found that dissociation of u-HMM heads
from pyrene-actin by ATP resulted in a
Fmax/Ffinal of ~0.4
in the absence of ADP and ~0.2 in the presence of ADP. We wondered why they did not observe a
Fmax/Ffinal of ~0.8
in the absence of ADP, as would be expected from previous studies with
tissue-derived smooth muscle S1 (28), and consequently ~0.4 in the
presence of ADP. A lower than expected pyrene-actin quenching by
expressed HMM might be due to the following. First, it could be an
inherent property of the molecule, although the amino acid sequence of the expressed HMM is identical to that of tissue-derived HMM except for
a FLAG tag at the C terminus. Second, it is possible that there are
unknown post-translational modifications specific to the tissue-derived
HMM. Third, a significant population of "dead heads" or "rigor
heads" (heads that bind irreversibly to actin) (21, 31) would lower
the
Fmax/Ffinal
without altering the observed stoichiometry. Berger et al.
(12) showed that ADP could dissociate ~40% of the heads from an
acto-u-HMM complex but not from an acto-tp-HMM complex. The rate of
this process for the acto-u-HMM complex was much slower than the
rate of ADP binding and thus was consistent with a
rearrangement. This suggested that these ADP heads initially bound to
actin but eventually found a thermodynamically more stable place to
bind or somehow lost their normal tight actin binding properties
(Kd < 40 nM). This result would be
obtained if dead heads were abundant, as we suggested previously, and
if the surface of dead heads had an extremely tight binding site for
the actin-binding site of a functional partner ADP head (perhaps in a
structure similar to that proposed by Wendt et al. (10,
11)). Formation of this nonphysiological structure might be expected to
be slow, as Berger et al. (12) observed, as it would involve
dissociation of a functional ADP head from actin followed by binding
(nearly irreversibly) to the partner dead head. The lack of such a
motor-motor domain interaction in a tp-HMM-ADP preparation
containing dead heads would be compatible with the structural data of
Wendt et al. (10, 11) showing no interaction between motor
domains in tp-HMM. Our results, under identical conditions to Berger
et al. (12), showed that ADP could not dissociate heads from
the acto-HMM complex regardless of the phosphorylation state (Table I).
Therefore, the results from both studies are internally consistent,
suggesting that the HMM preparations are different.
Berger et al. (12) reported that ~25% of u-HMM heads
bound ADP with an affinity of 2 µM, ~25% bound ADP
with a much weaker affinity, and the remaining ~50% did not respond
to ATP at maximal ADP concentrations. These nonresponsive heads may be
attributed to dead heads behaving as described above. Nevertheless,
their data strongly suggest that functional HMM binds to ADP with two different affinities. We are currently characterizing the ADP binding
properties of our preparations.
A test for the presence of dead heads is to compare the
Fmax/Finitial and
Fmax/Ffinal values for
association and dissociation experiments, respectively. These values
should be the same in the absence of dead heads. The
Fmax/Finitial from an
association experiment should not be affected by the presence of dead
heads, whereas the
Fmax/Ffinal from a
dissociation experiment would be lowered. In our study, both
dissociation (Table I) and association (Table II) of HMM heads from/to
pyrene-actin resulted in maximal fluorescence changes consistent with
earlier studies with tissue-derived smooth S1 (28). This agreement
between dissociation and association data is strong evidence that our
preparations do not contain a significant fraction of dead heads.
Furthermore, the single turnover measurements in Fig. 6 are consistent
with previous steady-state measurements from our laboratory (21). Dash
and Hackney (31) estimated that the V8-cleaved tissue-derived
preparation contains ~8% dead heads, consistent with the study of
Ellison et al. (21).
Our data do not rule out the possibility that the tissue-derived
u-HMM-ADP complex can adopt a conformation like that described by Wendt
et al. (10, 11). Indeed, our association rate data are
consistent with the idea that the heads interact in some manner in the
presence of ADP but not in its absence. Our association rate data are
in agreement with a previous study by Rosenfeld et al. (32).
They measured the rates of pyrene-actin binding of tissue-derived u-HMM
and tp-HMM with and without ADP at high actin/HMM ratios. The binding
was monophasic except for the u-HMM-ADP complex, which bound in a
biphasic manner with two phases of similar amplitude. They interpreted
these results to indicate that both heads of the u-HMM-ADP complex
bound to actin but that an interaction between the heads slowed the
binding of the second head. Under conditions similar to theirs (at the
highest actin/HMM ratios of Fig. 5), we made the same observations.
Therefore, our data, like those of Rosenfeld et al. (32),
are consistent with an interaction between the two heads of the
u-HMM-ADP complex, which must be broken to allow the second head to
bind to actin. It is possible that this interaction is between the two
motor domains, as described in the model of Wendt et al.
(10, 11), but our data do not address this structural issue. Our data
suggest that for tissue-derived HMM, if such an interaction is
occurring, it is not strong enough to compete with actin to prevent
binding of both heads to actin. We have also shown that one-headed
actin binding behavior for u-HMM-ADP is not a requirement for
down-regulation of smooth muscle myosin and that two-headed actin
binding in the presence of ADP is a property of the native, undamaged,
fully regulated molecule.