Temperature plasticity of contractile proteins in fish muscle
Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan
e-mail: awatabe{at}mail.ecc.u-tokyo.ac.jp
Accepted 13 May 2002
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Summary |
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Key words: actin-activated Mg2+-ATPase activity, activation energy, carp, Cyprinus carpio, chimeric myosin, Dictyostelium discoideum, inactivation rate constant, myosin, myosin heavy chain isoform, sliding velocity, subfragment-1, thermal acclimation, thermostability
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Introduction |
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One of the best examples of a trait that changes in an
acclimation-temperature-dependent manner at the level of the whole animal is
the maximum cruising speed. Goldfish acclimated to different experimental
temperatures show unique temperature/performance curves
(Fry and Hart, 1948). In
general, swimming speed increases at low temperatures and decreases at high
temperatures following cold-acclimation. The opposite responses are observed
following acclimation to warm temperatures. The mechanisms underlying such
changes in swimming performance have been shown to involve adaptations in the
activity and thermostability of myofibrillar ATPase
(Johnston et al., 1975
) as
well as alterations in force production and maximum contraction speed of
isolated muscle fibres (Johnston et al.,
1985
). It has been reported that changes in myofibrillar ATPase
activity following temperature transfer are apparent for carp after 1 or 2
weeks (Heap et al., 1985
).
However, a steady state is achieved after 4 or 5 weeks, but not in starved
individuals, suggesting that protein synthesis or the turnover of myofibrillar
component(s) is involved in the response.
This article shows that one of the myofibrillar proteins responsible for changes in muscle plasticity in association with temperature acclimation of carp is myosin, a major protein in the contractile apparatus. Furthermore, the structure/function relationship of a temperature-specific myosin isoform expressed in carp is described.
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Carp myosin isoforms are responsible for the temperature plasticity of fast skeletal muscle |
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Myosin was isolated from fast skeletal muscle of carp acclimated to 10 and
30°C for a minimum of 5 weeks and examined for its ATPase activities
(Hwang et al., 1990).
Ca2+-ATPase activity differed between myosins from cold- and
warm-acclimated carp, especially at KCl concentrations ranging from 0.1 to 0.2
mol l-1, when measured at pH 7.0. The highest activity at a
measuring temperature of 20°C was 0.32 µmol Pi
min-1 mg-1 at 0.1 mol l-1 KCl for
cold-acclimated carp and 0.47 µmol Pi min-1
mg-1 at 0.1 mol l-1 KCl for warm-acclimated fish.
Physiologically functional actin-activated myosin Mg2+-ATPase
activity differed markedly between cold- and warm-acclimated carp. The maximum
initial velocity (Vmax) at 20°C was 1.8 s-1
at pH 7.0 and 0.05 mol l-1 KCl for cold-acclimated carp, which was
1.6 times that for warm-acclimated carp
(Table 1). These differences
were in good agreement with those obtained with myofibrillar
Mg2+-ATPase activity for carp acclimated to both temperatures. No
differences were, however, observed in myosin affinity to actin
(Km). Differences in myosin properties between cold- and
warm-acclimated carp were further demonstrated by its thermal stability. The
inactivation rate constant (KD) of myosin
Ca2+-ATPase was 7.5x10-4s-1 at 30°C
and pH 7.0 for carp acclimated to 10°C, which was approximately twice that
for carp acclimated to 30°C (Fig.
1A), suggesting that myosin from carp acclimated to 10°C is
much more thermally unstable than myosin from carp acclimated to 30°C.
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Confirmation of changes in the myosin cross-bridge head, S1, in association with temperature acclimation of carp |
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Polyacrylamide gel electrophoresis in the presence of sodium pyrophosphate
(PPiPAGE) showed that carp acclimated to 10°C contained
four isoforms of chymotryptic S1 (Watabe
et al., 1994). Peptide mapping revealed that these consisted of
two types of S1 heavy chain, H1 and H2, with different primary structures.
Four S1 isoforms in total, H1(A1), H1(A2), H2(A1) and H2(A2), were separated
by PPiPAGE with two associated alkali light chains, A1 and
A2. Carp acclimated to 30°C contained another type of S1 heavy chain, H3,
and thus included two S1 isoforms, H3(A1) and H3(A2).
DEAE anion-exchange column chromatography separated these isoforms well,
revealing that S1 from carp consisted of three heavy chain isoforms with
molecular masses of 96 kDa (H1), 94 kDa (H2) and 92 kDa (H3)
(Guo et al., 1994). The
composition of these three S1 heavy chain isoforms in carp changed in
association with temperature acclimation. The H1 heavy chain was dominant in
10°C-acclimated carp and responsible for high actin-activated S1
Mg2+-ATPase activity and low thermostability. In contrast, the H3
heavy chain predominating in 30°C-acclimated carp showed low activity and
high thermostability. The H2 heavy chain was found in both 10°C- and
20°C-acclimated carp, but only at very low levels in 30°C-acclimated
carp. The H1 heavy chain made up approximately 55% of the total amount of the
three heavy chain isoforms in 10°C-acclimated carp, while the H3 heavy
chain made up approximately 85% of that in 30°C-acclimated carp
(Guo et al., 1994
). It is well
known that limited proteolysis of S1 with trypsin produces 25, 50 and 20 kDa
fragments in order from the N terminus
(Harrington and Rodgers,
1984
). The H3 heavy chain from 30°C-acclimated carp produced
the three fragments described above (Guo et
al., 1994
). However, the H1 heavy chain from 10°C-acclimated
carp produced a 23 kDa fragment in addition to these three fragments.
N-terminal amino acid sequence analysis suggested that the 23 kDa fragment
contained an N-terminal peptide normally found in the C-terminal region of the
50 kDa fragment.
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In vitro motility assay of carp myosin isoforms |
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These results suggest that the less thermostable, more flexible structure of myosin from cold-acclimated carp has a reduced activation energy for the contractile process, which allows the F-actin to slide fast over myosin filaments even at low temperatures. The present data have shown that the energy barrier for the sliding velocity of F-actin on the myosin from 10°C-acclimated carp is much lower than that on the myosin from 30°C-acclimated carp (Fig. 1B). This observation is consistent with the fact that the thermal stability of the myosin from 10°C-acclimated carp is reduced in comparison with that for the myosin from 30°C-acclimated carp (Fig. 1A).
There are numerous data suggesting that the dynamics of the structural
fluctuation of the protein molecule and its functional activities are closely
related (Huber, 1979;
Welch et al., 1982
;
Karplus and McCammon, 1983
;
Brooks et al., 1988
). It has
been observed that the less thermostable a protein, the more flexible its
structure (Delpierre et al.,
1983
; Wrba et al.,
1990
; Varley and Pain,
1991
). Varley and Pain
(1991
) have shown that, at a
given temperature, 3-phosphoglycerate kinase from thermophilic bacteria is
more stable, and the activation energy for the kinetic rate of acrylamide
quenching of tryptophan fluorescence is lower for the enzyme from mesophilic
yeast than for that from the thermophilic bacterium. In the myosin of
cold-acclimated carp, increased conformational dynamics resulting from lower
thermal stability would reflect the lower activation energy for the sliding
process. This low energy barrier for the sliding process would make it
possible for carp living at low temperatures to swim as the same speed as
those living at warmer temperatures.
Nakaya et al. (1995) have
reported that, in a differential scanning calorimetric study of a rod portion
prepared from the myosin of 10°C-acclimated carp, the myosin isoform was
less thermally stable than that from 30°C-acclimated carp. The differences
in thermal stability between the isoforms are therefore considered to span an
entire region of the myosin molecule, although the contribution of a given
region, for example S1, of the molecule to the changes in thermal stability
might vary. The activation energy of the actin-activated
Mg2+-ATPase was also lower in cold-acclimated carp myosin, although
the difference in Ea between the two carp myosin isoforms,
0.08 kJ mol-1, was smaller than the difference in
Ea for the sliding velocity, 48 kJ mol-1. The
difference in the activation energy between the sliding process and
actin-activated Mg2+-ATPase is consistent with the idea that the
rate process that limits sliding velocity is different from that limiting
ATPase activity (Siemankowski et al.,
1985
; Dantzig et al.,
1991
,
1992
). Furthermore, this
suggests that the conformational dynamics of a restricted region of myosin
governs the mechanical process and the conformational dynamics of the rest of
the myosin molecule governs the chemical process.
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cDNA cloning of myosin heavy chain isoforms from thermally acclimated carp |
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Rayment et al. (1993)
elucidated the three-dimensional structure of chicken pectoralis muscle S1,
showing that ATP is inserted into the cleft formed by the 50 kDa tryptic
fragment and located on the opposite side to the actin-binding region of the
same fragment. Myosin light chains are bound to the C-terminal region of the
-helical 20 kDa fragment. S1 contains two surface loops, loops 1 and 2.
Loop 1, connecting the 25 and 50 kDa tryptic fragments, is situated near the
ATP-binding sites, whereas loop 2 is situated on the actin-binding site in the
50 and 20 kDa fragments (Sutoh,
1982
,
1983
;
Chaussepied and Morales, 1988
;
Rayment et al., 1993
). It is
known that the two surface loops vary in both length and amino acid sequence
between different myosin types, including those from skeletal and smooth
muscle (Yanagisawa et al.,
1987
; Shohet et al.,
1989
; Maita et al.,
1991
; Bobkov et al.,
1996
).
The amino acid sequences of the entire myosin molecules were deduced from
DNA nucleotide sequences for the 10°C, intermediate and 30°C types.
The three isoforms generally resembled each other in primary structure,
showing 96.4, 93.8 and 93.6% identity between the 10°C and intermediate
types, between the 10 and 30°C types and between the intermediate and
30°C types, respectively. In contrast, S1 showed 95.0, 91.9 and 90.9%
identity, respectively, in these comparisons. However, isoform-specific
differences were clearly observed between the 10 and 30°C types in the
first 60 amino acid residues from the N terminus, where the intermediate type
was intermediate between the sequences of the 10 and 30°C types
(Hirayama and Watabe, 1997).
Another striking difference was observed in the two surface loops between the
10 and 30°C types. Five amino acid residues out of 16 were different in
loop 1 near the ATP-binding pocket, and six out of 20 were different in loop 2
on the actin-binding site (Fig.
2). Although the three types of carp S1 showed different amino
acid sequences in the two surface loops, both loops were of two skeletal
muscle types. The P-loop connecting the ß-sheets that are known to
surround the ATP-binding pocket had a highly conserved primary structure among
the three types. Amino acid substitutions between the 10 and 30°C types
are present not only in the above regions but also in a variety of areas in
the motor domain.
|
In northern blot analysis, the accumulated mRNA levels of the 10°C and
intermediate types were significantly higher in carp acclimated to 10°C
than in carp acclimated to 30°C, whereas the mRNA levels of the 30°C
type were significantly higher in carp acclimated to 30°C than in carp
acclimated to 10 and 20°C (Hirayama
and Watabe, 1997).
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Functional comparison of loops 1 and 2 of myosin S1 using chimeric myosin consisting of carp loops in a Dictyostelium discoideum myosin backbone |
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Actin-activated Mg2+-ATPase activity was measured for chimeric
myosins and for the Dictyostelium discoideum wild-type myosin at
20°C in the absence and presence of various concentrations of F-actin. The
two loop-1-associated chimeric myosins, loop 1-10 and loop 1-30, showed
Vmax and Km values similar to those of
Dictyostelium discoideum wild-type myosin
(Table 1). These results were
consistent with the data reported by Murphy and Spudich
(1998) for Dictyostelium
discoideum chimeric myosins containing loop 1 of rabbit skeletal or
Acanthamoeba myosin, which showed that chimeric substitutions of loop
1 did not affect the Vmax of actin-activated
Mg2+-ATPase activity. However, the Vmax of
actin-activated Mg2+-ATPase of the loop 2-10 myosin was 1.4 times
that of the loop 2-30 myosin, although the Km values for
actin were not significantly different
(Table 1). In contrast, all
chimeric myosins showed similar sliding velocities in in vitro
motility assay (Table 2). These
results were clearly different from previous findings that the loop 1
structure affected the sliding velocity of actin filaments
(Murphy and Spudich, 1998
;
Bobkov et al., 1996
;
Kelley et al., 1993
). As
described above, myosin prepared from carp acclimated to 10°C moved over
actin filaments faster than that from carp acclimated to 30°C. The
differences in motility of carp myosin isoforms are therefore probably caused
by amino acid substitutions in regions other than loops 1 and 2.
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Concluding remarks |
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Acknowledgments |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bobkov, A. A., Bobkova, E. A., Lin, S. H. and Reisler, E.
(1996). The role of surface loops (residues 204-216 and 627-646)
in the motor function of the myosin head. Proc. Natl. Acad. Sci.
USA 93,2285
-2289.
Brooks III, C. L., Karplus, M. and Pettitt, B. M. (1988). Proteins. New York: John Wiley & Sons.
Chaen, S., Nakaya, M., Guo, X. F. and Watabe, S. (1996). Lower activation energy for sliding velocity of F-actin on a less thermostable isoform of carp myosin. J. Biochem. 120,788 -791.[Abstract]
Chaussepied, P. and Morales, M. F. (1988). Modifying preselected sites on proteins: the stretch of residues 633-642 of the myosin heavy chain is part of the actin-binding site. Proc. Natl. Acad. Sci. USA 85,7471 -7475.[Abstract]
Dantzig, J. A., Goldman, Y. E., Millar, N. C., Lacktis, J. and Homsher, E. (1992). Reversal of the cross-bridge force-generating transition by photogeneration of phosphate in rabbit psoas muscle fibres. J. Physiol., Lond. 451,247 -278.[Abstract]
Dantzig, J. A., Hibberd, M. G., Trentham, D. R. and Goldman, Y. E. (1991). Cross-bridge kinetics in the presence of MgADP investigated by photolysis of caged ATP in rabbit psoas muscle fibres. J. Physiol., Lond. 432,639 -680.[Abstract]
Delpierre, M., Dopson, C. M., Selvarajah, S., Weldin, R. E. and Poulsen, F. M. (1983). Correlation of hydrogen exchange behaviour and thermal stability of lysozyme. J. Mol. Biol. 168,687 -692.[Medline]
Fry, F. E. J. and Hart, J. S. (1948). Cruising speed of goldfish in relation to water temperature. J. Fish. Res. Bd. Can. 7,169 -175.
Guo, X. F., Nakaya, M. and Watabe, S. (1994). Myosin subfragment-1 isoforms having different heavy chain structures from fast skeletal muscle of thermally acclimated carp. J. Biochem. 116,728 -735.[Abstract]
Harrington, W. F. and Rodgers, M. E. (1984). Myosin. Annu. Rev. Biochem. 53, 35-73.[Medline]
Hazel, J. R. and Prosser, C. L. (1974).
Molecular mechanisms of temperature compensation in poikilotherms.
Physiol. Rev. 54,620
-677.
Heap, S. P., Watt, P. W. and Goldspink, G. (1985). Consequences of thermal change on the myofibrillar ATPase of five freshwater teleosts. J. Fish Biol. 26,733 -738.
Hirayama, Y., Sutoh, K. and Watabe, S. (2000). Structurefunction relationships of the two surface loops of myosin heavy chain isoforms from thermally acclimated carp. Biochem. Biophys. Res. Commun. 269,237 -241.[Medline]
Hirayama, Y. and Watabe, S. (1997). Structural differences in the crossbridge head of temperature-associated myosin subfragment-1 isoforms from carp fast skeletal muscle. Eur. J. Biochem. 246,380 -387.[Abstract]
Huber, R. (1979). Conformational flexibility and its functional significance in some protein molecule. Trends Biochem. Sci. 4,271 -274.
Hwang, G. C., Ochiai, Y., Watabe, S. and Hashimoto, K. (1991). Changes of carp myosin subfragment-1 induced by temperature acclimation. J. Comp. Physiol. B 161,141 -146.
Hwang, G. C., Watabe, S. and Hashimoto, K. (1990). Changes in carp myosin ATPase induced by temperature acclimation. J. Comp. Physiol. B 160,233 -239.
Imai, J., Hirayama, Y., Kikuchi, K., Kakinuma, M. and Watabe,
S. (1997). cDNA cloning of myosin heavy chain isoforms from
carp fast skeletal muscle and their gene expression associated with
temperature acclimation. J. Exp. Biol.
200, 27-34.
Johnston, I. A., Divison, W. and Goldspink, G. (1975). Adaptation in Mg2+-activated myofibrillar ATPase activity induced by temperature acclimation. FEBS Lett. 50,293 -295.[Medline]
Johnston, I. A., Sidell, B. D. and Driedzic, W. R. (1985). Forcevelocity characteristics and metabolism of carp muscle fibres following temperature acclimation. J. Exp. Biol. 119,239 -249.[Abstract]
Karplus, M. and McCammon, J. A. (1983). Dynamics of proteins: elements and function. Annu. Rev. Biochem. 53,263 -300.
Kelley, C. A., Takahashi, M., Yu, J. H. and Adelstein, R. S.
(1993). An insert of seven amino acids confers functional
differences between smooth muscle myosins from the intestines and vasculature.
J. Biol. Chem. 268,12848
-12854.
Maita, T., Yajima, E., Nagata, S., Miyashita, T., Nakayama, S. and Matsuda, G. (1991). The primary structure of skeletal muscle myosin heavy chain. IV. Sequence of the rod and the complete 1,938-residue sequence of the heavy chain. J. Biochem. 110, 75-87.[Abstract]
Murphy, C. T. and Spudich, J. A. (1998). Dictyostelium myosin 25-50K loop substitutions specifically affect ADP release rates. Biochemistry 37,6738 -6744.[Medline]
Nakaya, M., Watabe, S. and Ooi, T. (1995). Differences in the thermal stability of acclimation temperature-associated types of carp myosin and its rod on differential scanning calorimetry. Biochemistry 34,3114 -3120.[Medline]
Rayment, I., Rypniewski, W. R., Schmidt-Bäse, K., Smith, R., Tomchick, D. R., Benning, M. M., Winkelmann, D. A., Wesenberg, G. and Holden, H. M. (1993). Three-dimensional structure of myosin subfragment-1: a molecular motor. Science 261, 50-58.[Medline]
Shohet, R. V., Conti, M. A., Kawamoto, S., Preston, Y. A., Brill, D. A. and Adelstein, R. S. (1989). Cloning of the cDNA encoding the myosin heavy chain of a vertebrate cellular myosin. Proc. Natl. Acad. Sci. USA 86,7726 -7730.[Abstract]
Siemankowski, R. F., Wiseman, M. O. and White, H. D. (1985). ADP dissociation from actomyosin subfragment 1 is sufficiently slow to limit the unloaded shortening velocity in vertebrate muscle. Proc. Natl. Acad. Sci. USA 82,658 -662.[Abstract]
Sutoh, K. (1982). An actin-binding site on the 20K fragment of myosin subfragment-1. Biochemistry 21,4800 -4804.[Medline]
Sutoh, K. (1983). Mapping of actin-binding sites on the heavy chain of myosin subfragment 1. Biochemistry 22,1579 -1585.[Medline]
Varley, P. G. and Pain, R. H. (1991). Relation between stability, dynamics and enzyme activity in 3-phosphoglycerate kinases from yeast and Thermus thermophilus. J. Mol. Biol. 220,531 -538.[Medline]
Watabe, S., Guo, X. F. and Hwang, G. C. (1994). Carp express specific isoforms of the myosin cross-bridge head, subfragment-1, in association with cold and warm temperature acclimation. J. Therm. Biol. 19,261 -268.
Watabe, S., Hwang, G. C., Nakaya, M., Guo, X. F. and Okamoto, Y. (1992). Fast skeletal myosin isoforms in thermally acclimated carp. J. Biochem. 111,113 -122.[Abstract]
Watabe, S., Imai, J., Nakaya, M., Hirayama, Y., Okamoto, Y., Masaki, H., Uozumi, T., Hirono, I. and Aoki, T. (1995). Temperature acclimation induces light meromyosin isoforms with different primary structures in carp fast skeletal muscle. Biochem. Biophys. Res. Commun. 208,118 -125.[Medline]
Welch, G. R., Somogyi, B. and Damjanovich, S. (1982). The role of protein fluctuations in enzyme action: a review. Prog. Biophys. Mol. Biol. 39,109 -146.[Medline]
Wrba, A., Schweiger, A., Schultes, V., Jaenicke, R. and Závodszky, P. (1990). Extremely thermostable D-glyceraldehyde-3-phosphate dehydrogenase from the eubacterium Thermotoga maritima. Biochemistry 29,7584 -7592.[Medline]
Yanagisawa, M., Hamada, Y., Katsuragawa, Y., Imanura, M., Mikawa, T. and Masaki, T. (1987). Complete primary structure of vertebrate smooth muscle myosin heavy chain deduced from its complementary DNA sequence. Implications on topography and function of myosin. J. Mol. Biol. 198,143 -157.[Medline]
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