From the
The myosin head (S1) consists of a wide, globular region that
contains the actin- and nucleotide-binding sites and an
The introduction of in vitro motility assays in the
1980s established that the myosin head (subfragment-1, S1)
A persistent aim in the motility
field has been to isolate a minimal fragment that can function as a
mechanochemical motor. In the case of kinesin, whose ATPase activity is
stimulated by microtubules, as few as 340 amino acids from the N
terminus are sufficient for enzymatic activity and for movement in a
motility assay (Stewart et al., 1993). An N-terminal 75-kDa
fragment from skeletal myosin was reported to have actin-activated
ATPase activity (Okamoto and Sekine, 1987), but subsequent attempts to
isolate such a proteolytic fragment proved unsuccessful. In light of
the x-ray structure of S1, it is clear that any fragment devoid of the
20-kDa region will lack activity, since the latter polypeptide extends
all the way from the C terminus of S1 to the nucleotide- and
actin-binding sites (Rayment et al., 1993). With the advent of
molecular genetics, it has been possible to produce a recombinant
fragment in Dictyostelium that contains only the globular
domain of S1 without the light chain binding region (Itakura et
al., 1993). This truncated head can generate force and movement in
a motility assay, although it displays very limited movement despite an
unusually high ATPase activity (Itakura et al., 1993).
Here, a truncated S1 was prepared by proteolytic digestion of
ELC-deficient myosin. We have shown recently that it is possible to
strip myosin of its light chains by gel filtration in the presence of
high concentrations of ammonium chloride (Lowey et al.,
1993a). Unmasking the heavy chain sequence that interacts with ELC
exposes sites susceptible to chymotrypsin, and thereby facilitates the
isolation of a globular domain from S1. This fragment shares the same
enzymatic activity as S1 prepared by papain or chymotrypsin, but its
ability to generate movement is considerably reduced compared to S1.
These studies support our earlier conclusion that the light
chain-binding domain is essential for efficient coupling of ATP
hydrolysis to movement.
Actin was prepared from chicken pectoralis acetone
powder (Pardee and Spudich, 1982) and stored at 4 °C as F-actin in
5 mM KCl, 5 mM imidazole, pH 7.5, 2 mM MgCl
Chicken RLC
was prepared essentially as described in Katoh and Lowey (1989), but
with further purification by ion-exchange chromatography. After
precipitation of RLC with 26% ethanol,
Myosin
without essential light chains (ELC-deficient myosin) was prepared as
follows: myosin (5 mg/ml) was incubated in 4.7 M NH
Chymotryptic S1 was
prepared from myosin under the same conditions as described above for
motor domain.
Papain S1 was prepared essentially as described in
Margossian and Lowey (1982). Myosin (10 mg/ml) in 0.2 M ammonium acetate, pH 7.2, 2 mM MgCl
Each of the subfragments was
further purified by gel filtration at RT on a Superose 12 column (1
Light
chain isoforms of S1 (S1A1 and S1A2) were separated by ion-exchange
chromatography on DEAE-Sephacel (Pharmacia). About 300 mg of
chymotryptic S1 in 30 mM NaCl, 50 mM imidazole, pH
7.5, 3 mM Na
Protein
concentrations were determined by measuring absorbance at 280 nm using
the following extinction coefficients (1 mg/ml): 0.55, 1.1, 0.75, 0.83,
and 0.5 for myosin, actin, chymotryptic S1, papain S1, and RLC,
respectively. The concentration of ELC-deficient myosin and motor
domain was determined by the Bradford method(1976) using myosin or
chymotryptic S1 as a standard.
In some
motility experiments, papain S1 was bound to the coverslip via
attachment to a anti-RLC monoclonal antibody (7C10; Winkelman and
Lowey, 1986) which was adsorbed to the nitrocellulose-coated coverslips
at 100 µg/ml. Alternatively, equimolar concentrations of papain S1
and antibody (15 µM of each) were incubated at 0 °C
for about 2 h, and applied at RT to a Superose 6 column (1
A proteolytic fragment (MD) encompassing the catalytic and
actin binding sites of S1, but devoid of the light chain binding
region, was prepared from ELC-deficient myosin. In principle, MD could
be prepared by simply digesting isolated myosin heavy chain free of all
LCs, but in practice the yield of MD is poor, probably due to the
tendency of free heavy chains to aggregate (Lowey et al.,
1993a). Alternatively, it has been shown that ELC can be removed from
chymotryptic S1 by ion-exchange chromatography at 37 °C
(Sivaramakrishnan and Burke, 1982), or by immunoadsorption in the
presence of 4.6 M NH
A recombinant fragment consisting of the motor domain has
been expressed in the cellular slime mold Dictyostelium discoideum (Itakura et al., 1993). The C terminus of this truncated
head, Asp-781 (numbered according to the adult chicken myosin sequence,
Maita et al., 1991), is only
An increase in enzymatic activity with increasing
truncation is reminiscent of the mechanochemical protein, kinesin, and
related members of this superfamily of microtubule-dependent motor
proteins (Stewart et al., 1993; Chandra et al.,
1993). Expressed Drosophila kinesin with as few as 340
N-terminal residues had a microtubule-stimulated ATPase rate that was
The motor domains from chicken myosin and Dictyostelium myosin both support gliding of actin filaments
by an in vitro motility assay, but the velocity is much slower
than observed for myosin. Dictyostelium MD moved actin at
In contrast to the Dictyostelium system, chicken S1 prepared by papain, which
contains both the RLC and the ELC, moved actin at an appreciably faster
rate (2.6 µm/s) than chicken MD (0.5 µm/s). Chymotryptic S1,
which lacks the RLC, moved actin at an intermediate velocity. The rate
for papain S1 could be increased to 4 µm/s when the S1 was attached
to the surface by means of an antibody directed against the N-terminal
region of the RLC (Winkelman and Lowey, 1986). It seems reasonable to
assume, therefore, that the movement of chicken MD would not increase
beyond 1 µm/s even if an attachment site were to be designed. This
rate for MD is remarkably similar to the rate of actin movement found
for myosin heavy chain free of all light chains (Lowey et al.,
1993a).
We conclude that the velocities for the chicken subfragments
are in qualitative agreement with the earlier results for chicken
myosins deficient in one or both light chains. It would seem that
independent of whether one removes light chain, and thereby exposes the
heavy chain to solvent, or whether one removes the light chain-binding
domain by proteolysis, the velocity of actin movement is roughly
proportional to the length of the neck region. A third approach in the
future will be to delete regions of the myosin light chain-binding
domain by molecular biology (Trybus, 1994). For the present, the
results are at least consistent with the hypothesis that the size of
the working stroke is closely coupled to the length of the neck domain
of the myosin molecule (Vibert and Cohen, 1988; Rayment et
al., 1993).
We thank David Morgan for assistance with computer
programs.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-helical,
extended region that is stabilized by the presence of two classes of
light chains. The essential light chain abuts the globular domain,
whereas the regulatory light chain lies near the head-rod junction of
myosin. Removal of the essential light chain by a mild denaturant
exposes the underlying heavy chain to proteolysis by chymotrypsin. The
cleaved fragment, or ``motor domain'' (MD), migrates as a
single band on SDS-polyacrylamide gel electrophoresis, with a slightly
greater mobility than S1 prepared by papain or chymotrypsin.
Three-dimensional image analysis of actin filaments decorated with MD
reveals a structure similar to S1, but shorter by an amount consistent
with the absence of a light chain-binding domain. The actin-activated
MgATPase activity of MD is similar to that of S1 in V
and K
. But the ability of MD to
move actin filaments in a motility assay is considerably reduced
relative to S1. We conclude that the globular, active site region of
the myosin head is a stable, independently folded domain with intrinsic
motor activity, but the coupling efficiency between ATP hydrolysis and
movement declines markedly as the light chain binding region is
truncated.
is sufficient to move actin filaments and generate force
(Toyoshima et al., 1987; Kishino and Yanagida, 1988). With the
publication of the crystal structure of S1 from chicken skeletal muscle
myosin (Rayment et al., 1993), it became clear that the myosin
head consists of distinct domains, which do not correspond to the three
fragments (25, 50, and 20-kDa) obtained by proteolytic digestion of
flexible loops in S1 (Mornet et al., 1989). Instead, there is
a globular domain containing the nucleotide- and actin-binding sites,
from which extends a long
-helix (the C-terminal half of the
20-kDa fragment) that is stabilized by the essential (ELC) and
regulatory light chains (RLC).
Protein Preparations
Chicken pectoralis myosin
was prepared as described in Margossian and Lowey(1982) and stored at
-20 °C in 0.6 M NaCl, 20 mM sodium
phosphate, pH 7.0, 1 mM DTT, 3 mM NaN,
50% glycerol.
, 3 mM NaN
.
120 mg of protein in 50
mM sodium phosphate, pH 6.2, 1 mM DTT, 3 mM
NaN
, was applied to a hydroxylapatite (Bio-Rad) column (1.5
20 cm) equilibrated with the same buffer, and eluted with a
50-500 mM sodium phosphate gradient (total volume
= 350 ml). Pooled fractions containing pure RLC were dialyzed
against 10 mM sodium phosphate, pH 7, 1 mM DTT and
freeze-dried in the presence of an equal weight of sucrose.
Cl, 50 mM sodium phosphate, pH 7.5, 1
mM MgATP, 0.1 mM EGTA, 8 mM DTT, 3 mM NaN
, at 4 °C for 10 min. Myosin heavy chain was
separated from dissociated light chains by applying 1 ml to a Superose
6 column (1.6
30 cm; FLPC, Pharmacia) at 21-22 °C,
equilibrated with 4.5 M NH
Cl, 50 mM sodium phosphate, 7.5, 2 mM EDTA, 0.5 mM ATP, 3
mM NaN
, at a flow rate of 1 ml/min. Inclusion of
ATP is essential to preserve the ATPase activity. Myosin heavy chain
was eluted in the void volume
30 min after protein application and
was collected on ice in the presence of 20 mM DTT, 2 mM MgATP. RLC (6 µM) was added immediately to myosin
heavy chain (2 µM heads) to stabilize the protein.
NH
Cl was removed by dialysis in the cold, first against 0.4 M NaCl, 5 mM MgCl
, 20 mM sodium
phosphate, pH 7.2, 1 mM DTT, 3 mM NaN
,
0.5 mM ATP (4-5 h), and then overnight against low salt
buffer (5 mM sodium phosphate, pH 6.2, 5 mM MgCl
, 3 mM NaN
, 1 mM
DTT, 0.5 mM ATP). Excess light chain was removed from
precipitated ELC-deficient myosin by centrifugation at 120,000
g for 30 min. The washed pellet was resuspended in a minimum
volume of high salt buffer (0.6 M NaCl, 20 mM sodium
phosphate, pH 7.5, 3 mM NaN
, 2 mM DTT)
and clarified at 150,000
g for 10 min (Beckman TL-100)
to remove aggregated material.
Myosin Subfragments
Motor domain was prepared by
proteolytic digestion of ELC-deficient myosin (4-5 mg/ml) in 0.12 M NaCl, 1 mM EDTA, 1 mM DTT, 20 mM imidazole, pH 7.0, 3 mM NaN with 30 µg/ml
chymotrypsin (Worthington Biomedical) for 15 min at RT, and stopped
with 2 mM phenylmethylsulfonyl fluoride (Sigma). Motor domain
was separated from insoluble material by centrifugation at 320,000
g for 20 min (Beckman TL-100).
, 1 mM DTT, was digested with 7 µg/ml papain (Cooper Biomedical) for
10 min at RT, and stopped with 2 mM
1-chloro-3-tosylamido-7-amino-L-2-heptanone (Sigma). Insoluble
material was removed by centrifugation at 320,000
g for 20 min (Beckman TL-100).
30 cm; FPLC, Pharmacia) equilibrated with 150 mM NaCl,
1 mM DTT, 3 mM NaN
, 10 mM sodium
phosphate, pH 7.2 (PBS), 0.1 mM ATP, at a flow rate of 0.5
ml/min. The order of elution on this column was papain S1, chymotrytic
S1, and motor domain, consistent with their expected sizes.
, 0.5 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride was applied to a column (2.5
30 cm) equilibrated in the same buffer and eluted with a 30-150
mM NaCl gradient (total volume = 800 ml). Pools
containing the fractionated isoforms were concentrated by dialysis
against saturated ammonium sulfate, pH 7.3, 10 mM EDTA. The
precipitated protein was collected by centrifugation, dialyzed againt
PBS, and was stored at -20 °C in 50% glycerol.
ATPase Measurements
The actin-activated MgATPase
activity was determined at 25 °C as described in Lowey et al. (1993b) except that the KCl and imidazole concentrations were
lowered to 5 mM each. Phosphate release was measured by the
colorimetric method of White(1982).
In Vitro Motility Assays
Either cleaned glass or
nitrocellulose-coated coverslips were used for the flow-through
microchamber. For motility on glass, coverslips were soaked overnight
in a solution containing 50% ethanol, 1.5 N HCl, washed
thoroughly with distilled water, and air-dried. The assay was performed
essentially as described in Warshaw et al.(1990). To remove
rigor heads, samples were spun 320,000 g for 20 min
(Beckman TL-100) with equimolar concentrations of actin in the presence
of MgATP just prior to the assay. Subfragments in PBS were perfused
through flow cells at 0.2-0.4 mg/ml. More concentrated samples
were necessary compared to myosin (50 µg/ml), in order to have
smooth movement. A higher percentage (
60%) of filaments moved on
glass than nitrocellulose when subfragments were used.
30
cm; FPLC) equilibrated with PBS, 0.1 mM ATP. The papain
S1
antibody complex was eluted in the void volume and applied to
cleaned glass for the assay. All motilities were performed at 30
°C.
Electron Microscopy and Image Analysis
Low-dose
(<1000 e/nm
) images of
filaments suspended in an unbroken sheet of negative stain (1% uranyl
acetate) over holes in a carbon support film where recorded at nominal
60,000
magnification on a Philips CM12 electron microscope. The
micrographs were digitized on a raster corresponding to about 0.75 nm
in the filaments using an Eikonix model 1412 scanner interfaced to a
Digital Equipment Corp. VAXstation 3100. Filament images used for
further processing were selected on the basis of uniform S1 or MD
binding, even staining, and the presence of straight or only gently
curved filaments. Images of curved filaments were straightened by
reinterpolation using a cubic spline function fitted to the filament
axis (Egelman, 1986). Fourier transform data extending to about 2.7 nm
axial and 2.0 nm radial resolution from about 20 images of each type
were averaged semiautomatically (Morgan and DeRosier, 1992), aligned to
a common origin, and used for three-dimensional helical reconstruction
by standard methods (cf. DeRosier and Moore, 1970).
``Difference maps'' were calculated in Fourier space between
each pair of reconstructed structures, and difference peaks that were
statistically significant at greater than the 99.5% confidence level (dashed lines in Fig. 4, D-F) were identified
by applying a Student's t test to the densities and
their variances (Milligan and Flicker, 1987).
Figure 4:
Density maps in projection of
reconstructed filaments. Acto-MD (A), acto-S1A1 (B),
and acto-S1A2 (C) in projection down the long strand n = 2 helices (``helical projections''). Acto-S1A1
and the statistically significant density difference (dashed
lines) between it and acto-MD (D); acto-S1A2 and the
difference between it and acto-MD (E); acto-S1A1 and the
difference between it and acto-S1A2 (F). The greater length of
the S1s is reflected in difference peaks 1 in D and E; the extra density close to actin (difference peak 2) is
present in D and F but not in E. Closer to
the helix axis, where reconstructions of helical structures are less
reliable due to their limited radial resolution, other difference peaks
occur at the positions of the outer actin domains in D and F, and probably represent residual scaling errors between the
maps.
Preparation of MD
Proteolytic digestion of
filamentous myosin has been routinely used to isolate S1 and the myosin
rod. Papain cleaves at Leu-842 resulting in an S1 that contains both
RLC and ELC, whereas chymotrypsin cleaves the heavy chain at Phe-814,
leading to a somewhat smaller S1 with just the ELC (reviewed in Lowey,
1994). A slight difference in mobility of the heavy chain for the two
S1 s is evident in SDS-polyacrylamide gel electrophoresis (Fig. 1, lanes 4 and 5). Until recently, it was
not possible to prepare a more truncated form of S1, but the ability to
strip the LCs from the heavy chain (lane 2) and reconstitute
the myosin with just RLC (LC2, lane 3) provided a method for
isolating the globular or motor domain (MD) of myosin (Lowey et
al., 1993a). The heavy chain has a potential cleavage site for
chymotrypsin at Phe-797 in the region where ELC normally binds. We
assume this residue is the C terminus of MD, but end group analysis is
needed to confirm the assignment. A small difference in mobility
between MD (lane 6) and chymotryptic S1 (lane 5) is
suggested by the gel pattern, but a difference in size of only 17
residues is difficult to detect. A slight displacement in elution
volume was also seen between MD and chymotryptic S1 by gel permeation
chromatography on Superose 12 (FPLC, data not shown). The preparation
of MD is very homogeneous as judged by the single electrophoretic band
on SDS gels, and a single symmetrical peak by gel filtration. The yield
from
10 mg of myosin was
1 mg.
Figure 1:
SDS-polyacrylamide gel electrophoresis
(12.5%) of myosin and its subfragments. Myosin (lane 1),
myosin depleted of light chains (lane 2), heavy chain
reconstituted with RLC (LC2, lane 3), papain S1 (lane 4), chymotryptic S1 (lane 5), and motor domain (lane 6). Note the slight increase in mobility of the
subfragment heavy chain as it is truncated and the absence of light
chains in the motor domain.
Characterization of MD by Electron
Microscopy
Actin filaments were decorated with MD in order to
obtain a low resolution picture of the MD structure. For comparison,
actin filaments were also decorated with the two light chain isoforms,
S1A1 and S1A2, the difference between them residing in the N-terminal
extension of the A1 light chain (Lowey, 1994). Visual inspection of the
negatively stained images in Fig. 2reveals the characteristic
arrowhead appearance of the complexes. The MD-decorated actin filament
looks significantly narrower, however, and the individual truncated
heads are more distinct than for S1-decorated filaments. Surface views
of reconstructed densities of these filaments show that all the head
fragments bind similarly to actin, but the S1 s are longer by virtue of
the ELC- (A1 or A2) binding domain (Fig. 3). There is also extra
density near the actin interface in the case of S1A1-decorated actin (panel B), which is not present in the surface view of
MD-decorated actin (panel A). These differences are more
clearly seen in density maps formed by projecting the density of the
reconstructed filaments onto a plane perpendicular to the helix axis (Fig. 4). The maps show that S1A1 and S1A2 have additional mass
at their C termini relative to MD (arrows marked 1 in panels D and E), which is consistent with the
presence of the ELC-binding site. Furthermore, the extra density near
the actin surface seen in S1A1 (arrow marked 2 in panel D), is also seen, but to a lesser extent, when comparing
S1A1 and S1A2 (panel F). This extra density has been seen
previously in three-dimensional maps of decorated filaments obtained by
cryo-electron microscopy and was ascribed to the N-terminal domain of
the A1 light chain (Milligan et al., 1990).
Figure 2:
Electron microscopy of F-actin decorated
with myosin head fragments. Electron micrographs of negatively stained
acto-MD (A and B), acto-S1A1 (C), and
acto-S1A2 (D), recorded under high dose (A) or low
dose conditions (B-D). The different width of the arrowhead complexes in B compared to C and D is best seen by viewing the filaments along their lengths at
a glancing angle. The high dose image (A) emphasizes the clear
separation of individual MD units bound to adjacent actin monomers. Bar, 50 nm.
Figure 3:
Surface views of reconstructed density
maps. Acto-MD (A), acto-S1A1 (B), and acto-S1A2 (C). The three myosin head fragments bind in a similar way to
actin, but the S1s are longer (region 1) and acto-S1A1 also
has extra density (region 2) close to the actin interface
which is absent from acto-MD. Region 2 is not as dense in acto-S1A2
(see Fig. 4C) and does not appear as a statistically
significant difference peak when acto-S1A2 and acto-MD are compared
(see Fig. 4E).
Functional Properties of MD
The actin-activated
MgATPase rate for MD was the same as for S1 prepared by papain or
chymotrypsin, within the experimental error (Fig. 5). The average V and K
for the
subfragments was about 20 s
and 60 µM,
respectively. These kinetic constants are well within the range of
values usually found for S1 (Margossian and Lowey, 1973; Wagner et
al., 1979). Removal of the light chain binding region does not
appear to affect the steady state ATPase activity of MD; whether
transient rates are affected remains to be determined.
Figure 5:
Actin-activated ATPase activity of myosin
subfragments. The activity was normalized to the value, 14.5
s obtained for chymotryptic S1 at 100 µM actin. Each rate is the average of three time points at any given
actin concentration, and rates were determined for several independent
preparations. V
extrapolated from
double-reciprocal plots was 30 s
for chymotryptic S1 (triangles), 22 s
for papain S1 (squares), and 18 s
for motor domain (circles). Note that the myosin from which motor domain was
prepared had been exposed to high concentrations of NH
Cl,
conditions which lead to a loss of about 20% activity (Lowey et
al., 1993a).
Despite a
constant ATPase activity, the subfragments showed marked differences in
their ability to move actin filaments in an in vitro motility
assay (Fig. 6). After purification by gel filtration, papain S1
showed the fastest movement, 2.6 µm/s; chymotryptic S1 was 1.5
µm/s; and MD was 0.5 µm/s. The motility depended to some extent
on the surface of the coverslip: the best movement was observed on
cleaned glass coverslips; fewer actin filaments moved on nitrocellulose
treated surfaces, although the rates of the filaments that did move
were not substantially different. In order to avoid unfavorable
interactions between S1 and the substratum, we first adsorbed the
nitrocellulose-coated coverslip with a monoclonal antibody (7C10)
specific for the N-terminal region of the RLC (Winkelmann and Lowey,
1986). The advantages of using monoclonal antibodies in the motility
assay for skeletal (Lowey et al., 1993a) and smooth muscle
myosin (Trybus et al., 1994; Trybus, 1994) have been described
previously. Attaching papain S1 by the antibody (7C10) interaction
increased the velocity to 4 µm/s; the same rate was obtained when
S1 and antibody were mixed in solution prior to adhesion to the
coverslip (Fig. 6).
Figure 6:
In vitro motility assay of myosin
subfragments. The subfragments: papain S1, chymotryptic S1, and motor
domain were applied directly to cleaned glass coverslips. Each velocity
value represents the average of a minimum of 40 filaments, and two
independent preparations (indicated by bars of different
shadings). In the case of (S1+Ab), papain S1 was either
attached to the coverslip via an antibody specific for the N terminus
of RLC (7C10), which was first adsorbed to a nitrocellulose-coated
coverslip (filled bar), or papain S1 was mixed with antibody
in solution and the complex after gel filtration was applied to the
coverslip (striped bar). All velocities are expressed as the
mean ± standard deviation.
Unfortunately, this type of attachment
cannot be used for chymotryptic S1 or MD, since antibodies binding
closer to the active sites will have an inhibitory effect on S1
movement (data not shown). A certain degree of uncertainty in motility
values is unavoidable when attaching head fragments to a surface, be it
by an antibody or by an engineered tag. However, it has been our
experience, and that of others (Stewart et al. 1993)), that if
a protein supports movement, the rate of movement will not change by
more than 2-fold independent of the mode of attachment to the
substratum.
Cl (Wagner and Giniger, 1981).
However, it is difficult to eliminate all traces of intact S1 by these
procedures, and, moreover, the isolated S1 heavy chain is relatively
unstable (Wagner and Stone, 1983). By using ELC-deficient myosin as the
starting material, it has been possible to overcome these difficulties
and prepare a homogeneous MD with good enzymatic activity and stability
over time.
16 residues shorter than
chicken motor domain. But the difference in enzymatic properties
between the two motor domains is striking: whereas the activity of
chicken MD is similar to that of S1 prepared by papain or chymotrypsin, Dictyostelium MD showed a >10-fold higher actin-activated
MgATPase than Dictyostelium S1. The latter was expressed with
both LCs and the heavy chain terminated at Pro-840 (Itakura et
al., 1993).
60-fold higher than the rate for bovine kinesin (Huang and Hackney,
1994). If the high ATPase rate for Dictyostelium MD is due to
truncation, it is not clear why chicken MD of similar length does not
have an elevated ATPase activity. Perhaps the region of the heavy chain
linking the MD to the light chain-binding domain is folded differently
in these two widely divergent species, and cleavage in this critical
linker region affects the active site of one myosin more than the
other. A comparison of the x-ray structure of the truncated Dictyostelium heavy chain (MD) with the three-dimensional
structure of chicken S1 has revealed remarkable conservation of
structure, except for differences in the C terminus of the heavy chain
following the reactive thiols (Rayment et al., 1995). No firm
conclusions can be drawn at the present time, however, since the Dictyostelium structure with its bound nucleotide and
truncated heavy chain is too different from chicken S1 for a direct
comparison.
0.12 µm/s compared to 1-2 µm/s for Dictyostelium myosin (25 °C, Itakura et al., 1993). Chicken MD
moved actin filaments at a rate of
0.55 µm/s compared to
7-8 µm/s for chicken myosin (30 °C). Therefore, there is
good agreement between the two species if one only considers the
relative rates of MD:myosin. There are difficulties, however, when
comparing the velocities of MD:S1. Dictyostelium S1 was able
to support actin gliding when bound directly to nitrocellulose (0.13
µm/s, Manstein et al., 1989), but Dictyostelium MD could not support movement until it was attached to the glass
surface via a biotin-avidin system (Itakura et al., 1993).
This fusion MD was now able to promote sliding motion, but the velocity
was the same as found earlier for Dictyostelium S1. Addition
of a biotin-avidin attachment site to the S1 did not increase its
velocity which remained at 0.13 µm/s.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.