From the Department of Physiology, University of
Wisconsin Medical School, Madison, Wisconsin 53706, § Department of Physiology, University of California School
of Medicine, Los Angeles, California 90095, and
¶ Department of Animal Science, University of Wisconsin,
Madison, Wisconsin 53706
Received for publication, July 19, 2000, and in revised form, November 10, 2000
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ABSTRACT |
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The kinetics of nucleotide turnover vary
considerably among isoforms of vertebrate type II myosin, possibly due
to differences in the rate of ADP release from the nucleotide binding
pocket. Current ideas about likely mechanisms by which ADP release is regulated have focused on the hyperflexible surface loops of myosin, i.e. loop 1 (ATPase loop) and loop 2 (actin binding loop).
In the present study, we investigated the kinetic properties of rat and
pig There is considerable interest in the sequence and subunit
composition of myosin isoforms due to the roles that variable
expression may have in determining the contractile properties of
myocardium and skeletal muscles. Type II myosin molecules are composed
of two heavy chains (MHC)1
(~220 kDa each) and two pairs of light chains (MLCs) designated the
essential (~25 kDa) and regulatory light chains (~16 kDa); both the
MHCs and MLCs derive from multi-gene families (for review, see Ref. 1).
The expression of different myosin subunit isoforms confers a wide
range of cross-bridge interaction kinetics in muscles of different
types and from different species, variations that are ultimately due to
divergence in the primary sequences of the encoding genes.
One feature common to all myosins is the presence of two surface loops
(loops 1 and 2) in the MHC subunit, but because of their
hyper-flexibility, these loops were not resolved in the x-ray structure
of chicken pectoralis myosin (2). Loop 1 is located in the vicinity of
the catalytic site, and loop 2 is close to the actin-binding site. The
sequences of the loops have been shown to vary significantly between
and within classes of myosins (3), and these variations are thought to
account for differences in the motile properties of these motor
proteins (4). Studies using chimeric Dictyostelium myosin
(5) and chimeric smooth muscle myosin (6) have shown that differences
in loop 2 alter the steady state ATPase activity, although it is still
not known whether loop 2 is a determinant of
Vmax, Km, or both. These
studies also show that the ATPase activities (in solution) and motor
velocities of myosins are not proportional, a finding that is
consistent with previous suggestions that actin-activated ATPase and
motility have different rate-limiting steps (7) and might therefore be
controlled by distinct molecular mechanisms.
The possible roles of loop 1 in conferring the kinetic properties of
myosin are not well understood. Spudich (4) proposes that loop 1 regulates the rate of ADP release, which would effectively control the
rate of actomyosin dissociation (which occurs almost immediately once
ADP dissociates as a result of rapid binding of ATP) and, therefore,
the mechanical velocity of the motor protein (8). The idea that loop 1 is a key element in the regulation of the nucleotide turnover rates in
myosin has been investigated using gizzard and vascular smooth muscle
MHC isoforms, which are spliced products of the same gene and differ in
a seven-amino acid insert in loop 1. Initially, Kelley et
al. (9) show that gizzard myosin (containing the 7-amino acid
insert) had twice the ATPase activity of aorta myosin and translocated
F-actin filaments 2.5-fold faster in an in vitro motility
assay. Consistent with these results, later studies showed that removal
of the insert halved both actin-activated ATPase activity and F-actin
sliding velocity (10). These kinetic effects of the loop 1 insert were subsequently related to the rate of ADP release, which was 3-fold faster in gizzard acto-S1 than in myosin without the insert (11). Furthermore, based on results from recombinant myosins with variable loop 1 sequences, Sweeney et al. (11) conclude that the size and the flexibility of loop 1 influence the rate of ADP release and
in vitro sliding speed, but that loop 1 is not the only
regulator of nucleotide turnover kinetics.
The heterogeneity observed in loop 1 and loop 2 sequences in the myosin
super-family (and among isoforms of type II muscle myosins) has been
taken as evidence that the loops have significant roles in regulating
the kinetic properties of motor proteins (3). Thus, it is tempting to
generalize previous conclusions (10, 11) and hypothesize that loop size
and flexibility modulate turnover kinetics throughout the class II
myosin superfamily. In the present study, we tested this idea by
performing a comparative analysis of contractile properties and
kinetics of nucleotide turnover in cardiac myosins with identical loop
1 sequences ( Experimental Animals
Rats--
Animal usage was conducted under strict guidelines
established by the University of Wisconsin Animal Care Committee. All
animals were housed in temperature- and light-controlled quarters and were provided with food and water ad libitum.
Thyroidectomized female Harlan Sprague-Dawley rats weighing 200-232 g
were purchased from Harlan Sprague-Dawley (Madison, WI).
Propylthiouracil (12 mg/kg) was administered daily by intraperitoneal
injection for 5 weeks to all thyroidectomized rats, since the
combination of thyroidectomy and supplemental propylthiouracil
administration has been shown to virtually eliminate circulating plasma
T3 and thyroxine (T4) (14). Hearts from these animals express virtually 100% Pigs--
Each pig (average weight of 134 kg) was electrically
stunned (280V, 60 Hz) before severing the carotid and jugular vessels. The hearts were then surgically removed. Hearts used for enzymatic preparation of cardiac myocytes for mechanical studies were immediately cannulated and perfused (see below) to prevent coagulation of blood.
All procedures were performed according to guidelines established by
the University of Wisconsin Animal Care Committee.
Other Species--
Tissue samples were also obtained from the
atria and ventricles of mice and rabbits using the methods described
above. A sample of dog ventricular myocardium was also obtained as a
gift from Dr. M. Wolff of the Section of Cardiology of the University
of Wisconsin. These samples were used to assess the variablity of homologous MLCs in mammalian hearts (see below).
Tissue Preparation
The left ventricle of each heart was carefully removed and
washed in oxygenated Ringer's solution. Transmural samples (~1 cm2) were removed from the left ventricular free walls of
pig hearts and rapidly frozen in liquid nitrogen. Samples used for
protein purification were subsequently placed on a pre-chilled mortar and ground under a constant stream of liquid nitrogen. Each sample was
placed in a chilled ( Additional hearts (rats, n = 8; pigs, n = 2) were enzymatically digested to obtain cardiac myocytes using a
method slightly modified from that of Strang et al. (15).
Each heart was quickly excised from the anesthetized animal, and the
aorta was cannulated for subsequent retrograde perfusion of the
coronary artery using a modified Langendorff apparatus. The hearts were
first perfused with a Ca2+-containing Ringer's solution
(1.2 mM MgCl2, 1.0 mM
CaCl2, 4.8 mM KCl, 118.0 mM NaCl,
2.0 mM, KH2PO4, 5.0 mM
pyruvate, 11 mM glucose, and 25.0 mM HEPES, pH
7.4). This was followed by successive perfusions with a
Ca2+-free Ringer's solution (4.5 min) and then with a
collagenase-containing (1.0 mg/ml) Ringer's solution (11 min) that
also contained hyaluronidase (1.0 mg/ml) and 0.05 mM
CaCl2. The ventricles were subsequently minced and
incubated for 20 min with the collagenase-containing Ringer's solution
plus 0.25% trypsin. Trypsin inhibitor (10 mg/ml; Sigma) was then added
to the solution. Undigested material was removed by filtration using a
300-µm Teflon mesh, and the cells were suspended in Ringer's
solution containing 1.0 mM Ca2+. All solutions
used in the isolation procedures were maintained at 37 °C and
bubbled with 100% O2. The isolated cells were then skinned
for 6 min at 22 °C in a solution containing 1.0 mM free Mg2+, 100 mM KCl, 2.0 mM EGTA, 4.0 mM ATP, 10 mM imidazole, pH 7.0, and 0.3%
Triton X-100. The skinned cells were finally washed in skinning
solution without Triton X-100 and stored at 0 °C for use on the same day.
Myosin Phenotypes
MHC phenotypes were assessed by SDS-PAGE using conventional
methods for the separation of cardiac isoforms (16) and with ultra-sensitive pulse-electrophoresis for SDS
gels.2 Phenotypes were
confirmed by Western blotting using a monoclonal antibody specific to
Amino Acid Sequences of Loop 1
Amino acid sequences for the two Unloaded Shortening Velocity (V0)
V0 was measured in maximally activated
myocytes using the slack test method as described by Strang et
al. (15). Once steady tension was achieved in activating solution,
the myocyte was slackened by 12-17% of initial length, corresponding
to a sarcomere length of 2.25 µm. The time between imposition of a
slack step and the redevelopment of force was measured by fitting a
horizontal line through the tension base line. The maximum slack
imposed was such that sarcomere length did not shorten below 1.90 µm,
which is about 0.1 µm greater than the length at which distortion due
to mechanical restoring forces within the myocytes is likely to occur (15). Length change (as percent initial length) was plotted versus duration of unloaded shortening (ms), and
V0 was determined from the slope of a line
fitted to the data by linear regression analysis. Data from a given
myocyte were discarded if the regression coefficient was <0.95.
Purification of Myosin and Generation of HMM
To purify myosin, the frozen muscle powder was gradually mixed
with a buffer (10.0 ml/g) consisting of 25 mM
Na2EGTA, 50 mM MOPS, 6.0 mM
magnesium acetate, 4.0 mM acetic acid, 5.5 mM
MgATP, 2 mM DTT, leupeptin (50 µg/ml), pepstatin A (7 µg/ml), and 0.2 mM phenylmethylsulfonyl fluoride at pH
7.1. The samples were stirred on ice for 5 min and then centrifuged at
10,000 × g for 10 min (4 °C) to pellet the
myofibrils. After discarding the supernatant, an aliquot of each sample
was removed for SDS-PAGE analysis, and the remaining pellet containing
the myofibrils was re-suspended in an extraction solution consisting of
0.3 M KCl, 10 mM
Na4P2O7, 1 mM
MgCl2, 5 mM EGTA, 150 mM
K2HPO4, 20 mM DTT, leupeptin (50 µg/ml), pepstatin A (7 µg/ml), and 0.2 mM
phenylmethylsulfonyl fluoride, pH 6.8. This suspension was stirred
constantly on ice for 90 min and then centrifuged for 20 min at
27,000 × g (4 °C) to remove unwanted cellular
debris. The supernatant was diluted in 20 volumes of ice-cold water
containing 1 mM DTT and allowed to stand for 2 h at
4 °C. The cloudy solution was centrifuged at 10,000 × g for 15 min, and the pellets were then resuspended in
solution containing 0.3 M KCl, 10 mM imidazole,
5 mM MgCl2, 20 mM DTT, 10 mM ATP, leupeptin (5 µg/ml), and pepstatin A (0.7 µg/ml), pH 6.8. The sample was gently homogenized and centrifuged in
a Beckman Optima TLX Ultrafuge at 480,000 × g for 10 min to pellet-contaminating actin. The supernatant was recovered,
diluted with 8 volumes of ice-cold water, and allowed to stand on ice for 3 h. The solution was then centrifuged at 27,000 × g for 10 min. The myosin pellet was re-dissolved in ~5 ml
of 0.6 M KCl, 2 mM MgCl2, 5 mM DTT, and 10 mM Tris, pH 7.6. An aliquot was
used for determination of protein concentration. The solution was then adjusted to a myosin concentration of 10 mg/ml, and digestion of HMM
was initiated by the addition of
1-chloro-3-tosylamido-7-amino-2-heptanone-treated chymotrypsin to 20 µg/ml. The sample was mixed and immediately transferred to a dialysis
slide (10,000-Da pore) and dialyzed with constant stirring at 4 °C
against 4 liters of 0.6 M KCl, 2 mM
MgCl2, 5 mM DTT, and 10 mM Tris, pH
7.6. This procedure was designed to remove contaminant nucleotide from
the samples. The chymotryptic digestion was stopped after 7 h by
the addition of phenylmethylsulfonyl fluoride to 1 mM. The
sample in its dialysis slide was then dialyzed for 2 × 4 h
against 4 liters of 25 mM MOPS, 2 mM
MgCl2, 2 mM DTT, 1 mM
K2EGTA, pH 7.3. This procedure precipitated undigested
myosin and light meromyosin and reduced any remaining free nucleotide
to <0.1 µM. Precipitation was typically observed within
15 min. HMM was recovered after a 10-min centrifugation at 480,000 × g using a bench-top centrifuge (Beckman Optima TLX Ultracentrifuge); KCl was then added to a final concentration of 100 mM to yield a total ionic strength of 120 mM.
Actin was prepared as described by Pardee and Spudich (21) from acetone
powder processed from the residue of rabbit muscle after myosin
extraction. A 120 µM stock of F-actin was dialyzed against 25 mM MOPS, 100 mM KCl, 2 mM MgCl2, 2 mM K2-EGTA,
2 mM DTT, pH 7.3.
Determinations of Protein Concentrations
The concentrations of pig and rat myosins and HMM were
determined from the absorbance at 280 nm using extinction coefficients (0.1%, mg Actin-activated Myosin-HMM ATPase
Actin-activated MgATPase activities were determined in low ionic
strength solutions, essentially as previously described (23). The
assays were conducted at 15 °C in 1-ml reaction volumes using constant concentrations of HMM and increasing concentrations of actin
from 0 to 25 µM. Inorganic phosphate generation was
measured at 3 or 4 time points after initiation of the reaction using a malachite green-based assay (24). Standard curves were prepared using
K2HPO4 standards. Linear regressions of
double-reciprocal plots were used to determine
Vmax and apparent Km.
Kinetics of ATP Binding, Cleavage, and ADP Dissociation
For transient kinetics, the rate of MgATP-induced dissociation
of acto-HMM and acto-HMM-ADP as well as cleavage rate were measured in
a micro-volume stopped-flow reaction analyzer SX.18MV (dead time of 1.6 ms) with Pro/Kineticist (Applied Photophysics). In all
experiments the temperature of the drive syringes, mixer, and
observation cell was regulated to 15 °C using a refrigerated water
bath. The ATP cleavage (hydrolysis) rate was estimated from the time
course of the HMM protein fluorescence increase after mixing with ATP.
Protein fluorescence was excited at 295 nm (using a grating
monochrometer with 1-mm slit width), and the change in protein
fluorescence was monitored at >320 nm (after passing through a WG
320-nm cut-off filter) using a photomultiplier as previously described
(25). The rates of dissociation of acto-HMM in the presence and absence
of MgADP were monitored by changes in light scattering at 340 nm (26).
MgATP-induced dissociation of acto-HMM was initiated by rapidly mixing
a solution of 4 µM actin, 2 µM HMM, and 1 µM AP5A (100 mM KCl, 25 mM MOPS, 1 mM EGTA, 1 mM
MgCl2, 1 mM DTT, pH 7.4) with one containing
0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, or 10.0 mM MgATP
(identical salts). To measure the rate of MgADP dissociation from
acto-HMM-MgATP, 40 µM or 180 µM MgADP was
added to the acto-HMM-AP5A solution before being mixed with
the same ATP concentrations as above. Exponential fits to the time
course of light scattering were performed using software routines
supplied by the stopped flow manufacturer. Typically the rates of 3-6
stopped flow records were averaged to obtain a rate representative of a
given MgATP concentration.
Myosin Phenotypes--
Gel analysis using conventional SDS-PAGE
and pulse-PAGE showed that the MHC content of myocardium from both pig
and hypothyroid rat ventricles were single protein bands with migration
mobilities characteristic of Loop Sequences--
The amino acid sequences of loop 1 were
obtained from the cDNA sequences for both rat and pig
The sequences were aligned to the numbering of the chicken
pectoralis MHC sequence (2) and include the nucleotide binding site
(designated the "phosphate loop") and loop 1 (residues 204-216). The results indicate that these regions are highly conserved between the Shortening Velocities--
Unloaded shortening velocities
(V0) were assessed at 15 °C in maximally
activated permeabilized myocytes from both pig and hypothyroid rat
ventricles (Table I). The procedure used
to attach the myocytes to the apparatus yielded consistently small end
compliances, since sarcomere length decreased by 3% or less during the
transition from rest to maximal activation. V0
was ~3-fold faster in rat left ventricular myocytes expressing
Actin-activated Myosin ATPase Activity--
The rates of substrate
utilization by rat and pig acto- Cleavage Rates--
The rates of ATP cleavage by rat and pig
Kinetics of Actomyosin-HMM Dissociation--
Estimates of the
kinetics of ATP binding to and ADP dissociation from rat and pig
Fig. 3B is a plot of the rate of AM dissociation as a
function of [MgATP]. As the rates of dissociation for the rat and pig preparations did not appear to differ significantly, the data were fit
by a single line. The solid line is a fit to the data of the following
form.
If ADP is added to an acto-HMM solution before mixing with MgATP, the
reaction shown in mechanism 2 (Equation 2) occurs. If the release of
ADP from acto-HMM, k+5, is much slower than the
rate of acto-HMM dissociation by ATP, i.e.
k+2K1[MgATP], then at
high concentrations of MgATP the rate of decrease in light scattering
will be limited by and equal to k+5. At small concentrations of MgATP (where k+5 > K1k2[MgATP]), MgADP reduces the kobs by competing with MgATP for
binding to AM and so decreases the fraction of A-HMM available to pass
through steps 1 and 2. Thus, at low [MgATP],
kobs will be small and will rise toward
k+5 as [MgATP] is increased. The rate of
acto-HMM dissociation was accurately fitted by a single rate of
dissociation for both pig and rat acto-HMM-ADP because the amount of
ADP added to the acto-HMM in each case was selected to produce a large
fraction of actomyosin as acto-HMM-MgADP before mixing. In addition,
there was significant ADP contamination of the pig acto-HMM, which
resulted from its 4-fold higher affinity for MgADP as compared with rat acto-HMM. To obtain an estimate of k Previous studies of the roles of loop 1 in smooth muscle
myosin isoforms showed that the peptide sequence of loop 1 is an important modulator of kinetic properties (9-11). If the peptide sequence of loop 1 is the primary determinant of the kinetic properties of muscle myosin isoforms, as has been suggested by Spudich (4), myosins having identical loop sequences would be expected to exhibit similar kinetic properties. In this study, we tested this hypothesis using pig and rat cardiac Several biochemical and physiological variables were used in this study
to assess differences in kinetics between rat and pig myosins
containing Stopped-flow measurements showed that the ADP off rate differed
~4-fold between rat and pig There have been some inconsistencies in previous studies regarding
possible roles of loop 1 in the actin-activated ATPase activity of
myosin. Results from recombinant Dictyostelium myosins suggested that Vmax is determined by the
sequence of loop 2 (5). Subsequent studies on recombinant smooth muscle
myosins showed that Km (not
Vmax) is determined by loop 2 (6) and that
Vmax is influenced by alterations in loop 1 (10,
11). In view of these earlier results, the sequences of loop 1 in the myosins used in the present study were also assessed. Our results show
that the peptide sequences of loop 1 were identical in rat and pig
Results obtained in this study also indicated that the rate of ATP
cleavage (k+h + k Overall, the results of this study support the interpretation that the
peptide sequence of loop 1 per se does not account for the
distinct kinetic properties of Much of our understanding of kinetic mechanisms in vertebrate myosins
has come from studies in smooth muscle myosins; however, conclusions
from smooth muscle myosins might not be easily extrapolated to cardiac
myosins. In this regard, smooth muscle myosin isoforms are spliced
products of the same gene and exhibit (at least in their HMM subunits)
variations only in loop 1, whereas in cardiac It is well recognized that tryptic digestion of HMM cleaves loop 1. However, the presence of nucleotide either in solution or cross-linked
to the active site of the protein offers protection against proteolysis
of the loop (36). This result suggests that loop 1 can assume two
different conformations, one in the absence and the other in the
presence of nucleotide in the active site (the latter making the
cleavage site less susceptible to proteolysis). All cardiac, skeletal,
and smooth muscle MHCs express charged consensus motifs at the
NH2-terminal ends of their respective loops (chicken
pectoralis numbering): cardiac
204DRSKKD219, skeletal
204EKKKE(E/D)209, smooth
204SHGKKD209. It is therefore conceivable that
such residues would mediate an interaction between loop 1 and another
region of the myosin backbone. Although we have yet to produce
recombinant proteins to test this idea, such a model would explain why
smooth muscle constructs (in which the only difference is the sequence
of loop 1) expressing a variety of foreign loops exhibited precisely
the same ATPase Vmax as the loopless constructs
(11). This model also provides an explanation for the positive
correlation between the ADP off rates and loop size among recombinants
(11); increasing the size of the loop might increase the probability of
interaction, if only to a small degree. Thus, we do not believe that
such a model conflicts with the results of Sweeney et al.
(11) regarding their myosin constructs having loops with lysine removal
or charge reversal, since these mutants retained charged residues in
the original positions of their consensus motifs. Finally, it should be
taken into account that loop 1 may not govern, directly or indirectly,
the ADP off rates of myosin (17) but, rather, other molecular steps.
-myosin heavy chains (
-MHC) in which we have found the sequences of loop 1 (residues 204-216) to be virtually identical, i.e. DQSKKDSQTPKG, with a single conservative substitution
(rat E210D pig). Pig myocardium normally expresses 100%
-MHC,
whereas rat myocardium was induced to express 100%
-MHC by surgical
thyroidectomy and subsequent treatment with propylthiouracil. Slack
test measurements at 15 °C yielded unloaded shortening velocities of
1.1 ± 0.8 muscle lengths/s in rat skinned ventricular myocytes
and 0.35 ± 0.05 muscle lengths/s in pig skinned myocytes.
Similarly, solution measurements at the same temperature showed that
actin-activated ATPase activity was 2.9-fold greater for rat
-myosin
than for pig
-myosin. Stopped-flow methods were then used to assess
the rates of acto-myosin dissociation by MgATP both in the presence and
absence of MgADP. Although the rates of MgATP-induced dissociation of
acto-heavy meromyosin (acto-HMM) were virtually identical for the two myosins, the rate of ADP dissociation was ~3.8-fold faster for rat
-myosin (135 s
1) than for pig
-myosin (35 s
1). ATP cleavage rates were
nearly 30% faster for rat
-myosin. Thus, whereas loop 1 appears
from other studies to be involved in nucleotide turnover in the pocket,
our results show that loop 1 does not account for large differences in
turnover kinetics in these two myosin isoforms. Instead, the
differences appear to be due to sequence differences in other parts of
the MHC backbone.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-MHC phenotype). The myosins were obtained from a small
animal (rat) and a large animal (pig), since any
species-dependent kinetic differences would likely be large
based on differences in animal size (12, 13). Because we find that
these myosins with identical loop 1 sequences have dramatically
different kinetic properties, we conclude that the sequence of the loop
does not confer unique kinetic properties to cardiac myosin nor is the
loop the only determinant of myosin turnover kinetics in cardiac
-MHC isoforms.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-MHC. Additional rats were made hyperthyroid to obtain hearts expressing 100%
-MHC, which was done by means of daily
intraperitoneal injections of levothyroxine (0.2 mg/kg) for 30 days.
Before organ collection, all animals were anesthetized in a glass bell
jar containing room air and 4% methoxyflurane. Deep anesthesia was confirmed by loss of reflexes and muscular tension. Animals were then
sacrificed by surgical introduction of pneumothorax.
80 °C) microcentrifuge tube and stored in
liquid nitrogen until analyzed. Samples from three animals of each
species were collected in sterile conditions and processed for RNA
extraction (see below) using a Polytron tissue homogenizer.
-MHC (219-1D1) (18). Characterization of the purity of
chymotrypsin-generated HMM was performed on 12-14% separating gels
(2% bisacrylamide), and MLC composition was assessed using either 16%
glycine or 14% Tricine separating gels. All gels were silver-stained
except for the HMM gels, which were stained with Coomassie Brilliant Blue.
-MHC isoforms were predicted
from cDNAs for the
-MHCs of rat and pig. RNA was isolated (19),
and its quality was assessed by agarose gel electrophoresis. cDNA
synthesis was performed with an RNase H-deficient reverse transcriptase
(ThermoscriptTM RT, Life Technologies, Inc.) according to
the manufacturer's instructions using an oligo(dt) (20) and another
reverse primer (5'-gtgatggccttgaaccg-3') that is complementary to exon
36 of all mammalian class II MHCs (20). Polymerase chain reaction was
performed using the Platinum DNA polymerase system (Life Technologies), also according to manufacturer's instructions, using the following primers: reverse for both rabbit and pig
(5'-cagctgctgcttgtcgttctccaggtccat-3'); forward for rat
(5'-tcagtcatggcggatggagagatggct-3') and forward for pig
(5'-aaggcggcatggtagatgcggagatg-3'). Double-stranded polymerase chain
reaction products (3.2 kilobases) were cloned into plasmid vectors
(Zero Blunt, Invitrogen) and sequenced (5'-gtgcgtggagcgcagctttct-3'; 5'-cacagtcgtctctccctggga-3'; 5'-gtgatggccttgaaccg-3').
1 cm
1) of
0.64. The concentration of rabbit actin was determined using an
extinction coefficient of 0.66 at 290 nm (22). The molecular masses
(g/mol) used for the calculation of molar concentrations of protein
species was 350,000 for HMM and 43,000 for actin. The concentrations of
nucleotides (ATP and ADP and AP5A) were calculated using
extinction coefficients at 259 nm.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-MHC (Fig.
1A). Western blots confirmed
that both pig and hypothyroid rat ventricles expressed 100%
-MHC, i.e. there was no immunoreactivity of tissues with an
anti-
MHC monoclonal antibody (results not shown) and positive
immunoreactivity with an anti-
MHC monoclonal antibody (Fig.
1B). Euthyroid rats exhibited positive immunoreactivity to
both monoclonal antibodies. Pulse-PAGE also showed that the migrations
of rat and pig
-MHCs were similar but not identical, suggesting that
these two motor proteins have some differences in primary sequence.
Samples from hyperthyroid rats exhibited a single protein band
corresponding to
-MHC (Fig. 1A).
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Fig. 1.
Characterization of MHC phenotypes of rat and
pig left ventricular myocardium. A, upper
panel, standard 12% SDS-PAGE. Lane 1, MHC composition
in euthyroid rats (fastest migrating band corresponds to -MHC).
Lane 2, MHC composition in hypothyroid rats. Lane
3, MHC composition in euthyroid pig. Middle panel,
pulse-PAGE in 12% SDS gels, which were run for 78 h. Lane
1, MHC composition in euthyroid rats (fastest migrating band
corresponds to
-MHC and the two upper bands correspond to two
-MHC isoforms). Lane 2, MHC composition in hypothyroid
rat. Lane 3, MHC composition in euthyroid pig. Lower
panel, Western blot analysis of MHC composition using anti-
MHC
monoclonal antibody. Lane 2, hypothyroid rat. Lane
3, euthyroid pig. Both samples exhibit positive immunoreactivity.
B, resolution of the MLC composition of left ventricular
myocardium of different species in 14% Tricine SDS-PAGE. From left to
right: Pg, pig; Dg, dog; Rb, rabbit;
Ms, mouse; Rt, rat.
-MHCs. The
sequences rat and pig
-MHC were, respectively, as follows.
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Sequence I
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[in a new window]
Sequence II
-MHCs of rat and pig ventricles and that the size, flexibility, and charge density of loop 1 are identical for the two motor proteins, i.e. there is a single conservative substitution, E209D.
-MHC (1.1 ± 0.8 ML/s; mean ± S.D., n = 8) than in pig left ventricular myocytes (0.35 ± 0.05 ML/s;
mean ± S.D., n = 5). Control experiments on left
ventricular myocytes from euthyroid rats showed that
V0 in myocytes expressing 100%
-MHC was
~2.3-fold faster (2.5 ± 1.2 ML/s; n = 10) than for rat myocytes expressing 100%
-MHC.
Measured kinetic constants
-HMMs were assessed by measuring
actin-activated ATPase activity at low ionic strength and 15 °C. The
ATPase rates for both motor proteins were hyperbolic with respect to
actin concentration and exhibited saturation kinetics at high actin
concentrations, which allowed us to use double-reciprocal plots to
estimate both Vmax and Km
(Table I). While Vmax of the ATPase was 2.9-fold greater in rat than in pig
-HMM, the Km for the
two motor proteins differed by just 1.5-fold.
-HMMs were estimated from the time course of protein fluorescence
increase observed after mixing with MgATP (25). The measurements were
performed at 15 °C, 120 mM ionic strength, and pH 7.4. In these studies we assumed that the reaction mechanism was similar to
that deduced by Johnson and Taylor (25) as given by reaction mechanism
1 below
where M is myosin. Step c is a diffusion-limited
collision reaction, step i is a fast (ki > 1000 s
(Eq. 1)
1) essentially irreversible
isomerization, and step h is a readily reversible
hydrolysis step where ** indicates an increased protein fluorescence. All the samples exhibited a typical fluorescence enhancement of about 5% upon mixing of MgATP with HMM (Fig.
2A), and the rate of
fluorescence increase rose with increases in [MgATP] (Fig.
3A). All traces were well fit
with a single exponential, and plots of the observed rates
(kobs) versus increasing [MgATP] (Fig. 3A) yielded rates that rose toward an asymptote equal
to (k+h + k
h) (25, 27). When [ATP] is small,
Kc[ATP]
1 and kobs
(k+h + k
h), so that
kobs = Kcki[ATP]. Thus, linear
regression analysis of kobs versus
[MgATP] for values less then 50 µM yielded a slope
corresponding to Kcki. These
plots indicated that Kcki for
porcine cardiac
-HMM was 3.3 × 105 M
1 s
1, and for
rat
-HMM was 6.0 × 105 M
1 s
1. These
values are in good agreement with earlier work using cardiac S-1 (27).
The value of (k+h + k
h) was approximated by progressively
increasing the [MgATP] until kobs no longer
changed. The values of kobs at 1 and 2.5 mM MgATP were not significantly different in either of the
HMMs tested so that (k+h + k
h) was computed from the average
kobs at these two concentrations. Thus
(k+h + k
h) for rat and pig
-HMM were
63.5 ± 1.4 s
1 and 43.5 ± 0.7 s
1 (n = 8), respectively.
These results are consistent with the remarkably similar structures of
the two myosins and with results from earlier studies (27).
View larger version (15K):
[in a new window]
Fig. 2.
Stopped flow measurements of HMM fluorescence
increase, the dissociation of actomyosin-HMM, and the dissociation of
actomyosin-HMM-ADP by MgATP. Panel A shows the time
course of the intrinsic fluorescence increase marking the ATP
hydrolysis by rat (a) and pig (b) HMM at 100 µM MgATP. The rate of the fluorescence increase in trace
a is 43.0 ± 0.4 s 1 and in
trace b is 35.9 ± 0.3 s
1.
Panel B shows the decrease in light scattering by acto-HMM
in rat (a) and pig (b) acto HMM in 250 µM MgATP. Trace a was fitted by a single
exponential whose rate was 330.7 ± 7.7 s-1 (amplitude
3.17 V), and trace b was fitted by a double exponential
equation whose rates were 392.8 ± 25.7 s
1 (amplitude = 4.31 V) and 8.2 ± 4.9 s
1(amplitude = 2.5 V). Panel
C shows the dissociation of rat (a) and pig
(b) acto-HMM-ADP in the presence of 2.5 mM
MgATP. Traces a and b were fitted with single
exponentials with rates of 135.5 ± 0.4 s
1 and 38.7+ 0.3 s
1, respectively. In each case,
r2 > 0.97. Each reaction time course is the
average of 3-4 separate recordings. The solid smooth lines
are exponential fits to the data with the indicated rates.
View larger version (17K):
[in a new window]
Fig. 3.
ATP dependence of the rates of intrinsic
protein fluorescence increase, acto-HMM dissociation, and acto-HMM-ADP
dissociation. Panel A shows the increase in intrinsic
fluorescence for rat (a) and pig (b) -MHC.
Panel B is the rate of acto-HMM dissociation in the absence
of ADP for rat (a) and pig (b) acto-HMM.
Panel C is the rate of acto-HMM dissociation in the presence
of MgADP for rat (a) and pig (b) acto-
-HMM. In
the experiments using rat
-HMM, the [MgADP] was 90 µM and for pig was
-HMM was 25 µM (20 µM added and 5 µM contamination). The
solid lines are fits to the data described in the text. In
each case, filled circles represent data from experiments
using rat
-HMM and filled circles for pig
-HMM. The
error bars represent ± 1 S.E.
cardiac acto-HMMs were obtained by measuring the
ATP-dependent rate of acto-HMM dissociation in the presence and absence of added ADP. The use of ADP as a competitive inhibitor of
ATP binding at the active site allows estimation of ADP off rates (26).
The reaction for the increase in light scattering is given in mechanism
2 below
where A is actin, M is myosin, and AM is actomyosin. In the
absence of ADP, only steps 1 and 2 occur when acto-HMM is rapidly mixed
with MgATP. Fig. 2B shows the decrease in light scattering upon mixing MgATP with acto-HMM (15 °C and 120 mM ionic
strength), which effectively reports dissociation of acto-HMM. The data
for rat acto-HMM were well fit using a single exponential, which was the case for 8 of 17 different conditions measured. When double exponential fits were made, the fast phase had a similar rate to that
fitted by a single exponential, and the second phase amplitude averaged
7.3% that of the fast phase. The data for pig acto-HMM dissociation
were also well fit (mean r2 = 0.983), with a
single exponential in 13 of the 31 conditions studied. However, a
double exponential yielded a better fit in the remainder of the
experiments. In these cases the amplitude of the second phase averaged
26.0 ± 3.0% (±S.E.) of the total change in light scattering,
and its rate was <35 s
(Eq. 2)
1, whereas that of the
fast phase was 2-20 times faster. As [MgATP] was increased, the
slower second phase rate increased relatively little, whereas the first
phase became much faster. As [MgATP] increased, the observed
amplitude of the first phase declined because an increasing fraction of
the phase occurred within the dead time of the stopped-flow device.
Thus, determinations of observed rates greater than 500-600
s
1 were less accurate, as fewer data points
were available for fitting. Similar two-phased behavior has previously
been observed in cardiac myosin, and the slower, less ATP-sensitive
rate has been attributed to denatured protein (27). However, we
conclude that the slower phase was produced by ADP contamination of our
pig acto-HMM preparation. This conclusion follows from our observation
that treatment of a pig acto-HMM preparation with 4 units of apyrase/ml
acto-HMM for 2 h before stopped-flow experimentation abolished the
second slow phase but did not change the fast ATP-dependent
rate. Thus, this contamination does not materially affect the
conclusions from these experiments.
This fit yielded
k2K1 of 1.7 ± 0.24 × 106 M
(Eq. 3)
1
s
1 and a K1 value of
904 ± 444 M
1. These results
imply that k2 is 1880 s
1 for rat and porcine
-HMMs. The error in
K1 is large because accurate measurements at
higher [ATP] were not possible.
5, we used
the value of K1k+2 from the data in Fig.
3B and the asymptote in Fig. 3C, a for
k+5 in Equation 2. Using
K1k+2 and
k+5, we computed the rates of rat acto-HMM-MgADP
dissociation using simulations of Equation 2 and found that the rates
observed at different [MgATP] were predicted if
k
5 = 3 × 106
M
1 s
1
(the solid lines in Fig. 3C, a are the
predicted rates). We then assumed the same k
5
for pig acto-HMM and found that the predicted dissociation rates were
accurately predicted by the equation (see solid line in Fig.
3C, b) if we assumed that there was 10 µM contaminating Mg-ADP in the pig acto-HMM before the addition of MgADP. The results of the fitting process are presented in
Table I. Because the length and sequence of loop 1 are essentially identical for the two species and the rate of ADP release from acto-rat
-HMM is about four times faster than that for acto-pig
-HMM, we
conclude that the rate is not controlled by loop 1. Furthermore, the
larger rate of ADP release from acto-rat-
-HMM is consistent with the
differences observed in unloaded shortening velocity measurements in
skinned myocardial preparations (Table I).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
myosins, which exhibit no differences in
loop 1 sequence (see "Results"). Contrary to expectations, our
results show that kinetic properties differ markedly between these two
cardiac myosins.
-MHC. Unloaded shortening velocity, which varies with the
rate of cross-bridge detachment (28), was used as an indirect assay of
ADP off rates (for review, see Ref. 29). Rat myocytes expressing
-MHC were used as controls and exhibited shortening velocities that
were 2.2-fold faster than rat myocytes expressing
-MHC, which is
consistent with previous results from cardiac myocytes (15) and from
purified myosin in an in vitro motility assay (30).
Remarkably, there was a 3-fold difference in V0
between rat
-MHC and pig
-MHC myocytes measured under identical
conditions (Table I). Although these results strongly suggest that
there are marked differences in kinetics between the two
-MHC motor
proteins studied here, such results are not entirely definitive because
they were obtained from preparations with intact myofilaments, which in
rat and pig myocytes contain different regulatory proteins.
Consequently, more direct kinetic measurements were performed using HMM
generated from purified myosins from both pig and hypothyroid rat myocardium.
-HMM myosins. These results indicate that there is good proportionality between mechanical measurements of
V0 and the rates of ADP dissociation, which is
consistent with the idea that ADP off rate is the primary determinant
of V0 (8). It seems likely that the variations
in
myosin heavy chain account for the differences in cross-bridge
kinetics observed in this study. A potential criticism of this
conclusion is that V0 is also modulated by MLC
content, at least in skeletal muscle, and it is possible that pig and
rat myosins do not have identical MLC content. For example, significant
depression of shortening velocity (or F-actin sliding velocity in
vitro) has been reported in MLC-deficient myosins (31, 32) and in
LC-reconstituted skeletal muscle myosins expressing greater amounts of
MLC 1f than MLC 3f (33, 34). It is unlikely that any of the myosins in our studies was LC-deficient, since MHC:MLC stoichiometry was similar
in all of our SDS gel samples (results not shown). Another possibility
that is difficult to eliminate completely is that there are differences
in the sequences of rat and pig cardiac MLCs. In this regard, the
sequences for pig cardiac MLCs are still unknown, making it impossible
to identify amino acid substitutions that might have significant
effects on function. At a superficial level, both pig and rat left
ventricular myocardium express MLC 1v and MLC 2v isoforms that migrate
similarly on SDS-gels, suggesting that the isoforms are likely to be
highly homologous in the two species (Fig. 1B). To date,
investigations of possible effects of altered MLC composition in the
mammalian heart has been done in only a few studies. However, mouse
transgenic studies appear to indicate that remodeling the MLC isoform
expression in the heart, at least for the regulatory MLC, does not
produce large changes in kinetics. For example, partial replacement of
MLC 2v with skeletal fast isoform (MLC 2f) does not alter
V0 in myocardium, even though it reduces ATPase
activity by about 20% (35). In the present study, we observed near
proportionality between V0 and the rate of ADP
dissociation as well as with the rate of actin-activated ATPase for the
-MHC motor proteins studied here. Although it is possible that
differences in MLCs influenced the kinetic properties of these motor
proteins, it is improbable that such differences could account for the
3-4-fold differences in kinetic properties observed between the rat
and pig isoforms of
-MHC.
-MHCs, and yet Vmax was 2.9-fold faster for
rat
-HMM than for the pig. Obviously, loop 1 cannot account for the
kinetic differences between these two heavy chains. Similarly, it must be concluded that loop 1 does not directly regulate mechanical or
biochemical Vmax in cardiac myosins.
Furthermore, the Km of the ATPase differed for the
two
-MHCs, although the difference (1.5-fold) was approximately half
that observed for Vmax. This disparity in degree
of difference for these variables in the two
-MHCs suggests that
different domains of the molecule are involved in setting these
variables. In this regard, it should be noted that the loop 2 sequences
of rat
-MHC and pig
-MHC exhibit one single nonconserved
substitution,3
i.e. rat A631T pig, according to the chicken pectoralis
numbering scheme (2).
h) was 30% faster in rat (~64
s
1) than in pig (~44
s
1)
-HMM myosins. Similarly, the binding
constants (Kcki) for ATP were
somewhat greater for rat (6.00 ± 0.03 × 105
M
1 s
1)
than for pig (2.70 ± 0.01 × 105
M
1 s
1),
suggesting that these properties of myosin are not directly related to
the sequence of loop 1.
-MHCs from different mammalian species and do not support the hypothesis proposed by Spudich (4)
concerning possible roles of loop 1. Thus, other regions or domains of
the myosin molecule must ultimately govern the kinetic properties
studied here. Amino acid sequence comparisons between rat and pig
-MHCs using both published sequences (GenBankTM
accession numbers AAB37320 for pig
-MHC and S06006 for rat
-MHC)
and our own results4 indicate
that there are regions of divergence, localized in three clusters: 1) 5 substitutions between residues 323 and 351, which forms an
-helix
close to the catalytic domain, in proximity to switch 1 and switch 2 and extending to the actin binding site; 2) 3 substitutions between
residues 423 and 432, located at the binding interface with actin; and
3) 4 substitutions between 575 and 612, also at the myosin-actin
binding interface. Amino acid substitutions in each of these regions
have been associated with familial hypertrophic cardiomyopathy, and it
seems likely that the kinetic differences observed in the present study
are due to one or more of these sequence differences. Our observations are consistent with the previous interpretation (11) that loop 1 is not
the primary determinant of kinetic differences between myosin isoforms.
Certainly, our results do not eliminate the possibility that loop 1 is
involved in regulation of myosin kinetics. From the results of Sweeney
et al. (11), in which removal of the loop virtually stopped
ADP release from the pocket, it appears that the loop is at least
necessary for nucleotide turnover. Thus, it is reasonable to speculate
about the role of loop 1 in myosin function.
myosins, the sequence
of the loop 1 is identical but the backbone varies.4
Because some of the substitutions in these
myosins are close to the
active site, it is reasonable to assume that their kinetics are
primarily determined by residues in the catalytic domain. In such a
case, the role of loop 1 would be to somehow influence, either to
hinder or promote, catalytic events within the binding pocket. For this
to occur, loop 1 would have to interact with sites in the backbone of
the molecule that directly govern the kinetic properties of the motor protein.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Kevin Strang and Mike Simek for assistance in performing V0 measurements in cardiac myocytes.
![]() |
FOOTNOTES |
---|
* This study was sponsored by National Institutes of Health Grants HL61635 (to R. M.) and 30988 (to E. H.) and by Portuguese Foundation for Science and Technology Grant BPD/1236/98 (to J. S. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Published, JBC Papers in Press, November 13, 2000, DOI 10.1074/jbc.M006441200
To whom correspondence should be addressed. Tel.:
608-262-1939; Fax: 608-265-5072; E-mail:
rlmoss@physiology.wisc.edu.
2 J. A. A. Sant'Ana Pereira, M. L. Greaser, and R. L. Moss, submitted for publication.
3 J. Sant'Ana Pereira, personal observations.
4 J. Sant'Ana Periera, D. Pavlov, M. Nili, M. Greaser, E. Homsher, and R. Moss, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: MHC, myosin heavy chain; MLC, myosin light chain; AP5A, P1,P5-di(adenosine 5')-pentaphosphate; PAGE, polyacrylamide gel electrophoresis; HMM, heavy meromyosin; MOPS, 4-morpholinepropanesulfonic acid; DTT, dithiothreitol; ML, muscle lengths; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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