Changes in carp myosin ATPase induced by temperature acclimation

Summary Myosins were isolated from dorsal ordinary muscles of carp acclimated to 10°C and 30°C for a minimum of 5 weeks and examined for their ATPase activities. Ca²⁺-ATPase activity was different between myosins from cold-and warm-acclimated carp, especially at KCl concentrations ranging from 0.1 to 0.2 M, when measured at pH 7.0. The highest activity was 0.32 mol P_i·min^-1·mg^-1 at 0.2 M KCl for cold-acclimated carp and 0.47 mol P_i·min^-1·mg^-1 at 0.1 M KCl for warm-acclimated fish. The pH-dependency of Ca²⁺-ATPase activity at 0.5 M KCl for both carp was, however, similar exhibiting two maxima around 0.3 mol P_i·min^-1·mg^-1 at pH 6 and 0.4 mol P_i·min^-1·mg^-1 at pH 9. K⁺(EDTA)-ATPase activity at pH 7.0 neither exhibited differences between both myosins. It increased with increasing KCl concentration showing the highest value of about 0.4 mol P_i·min^-1·mg^-1 at 0.6–0.7 M KCl. Actin-activated myosin Mg²⁺-ATPase activity was markedly different between cold-and warm-acclimated carp. The maximum initial velocity was 0.53 mol P_i·min^-1·mg^-1 myosin at pH 7.0 and 0.05 M KCl for cold-acclimated carp, which was 1.6 times as high as that for warm-acclimated carp. These differences were in good agreement with those obtained with myofibrillar Mg²⁺-ATPase activity between both carp. No differences were, however, observed in myosin affinity to actin. Differences in myosin properties between cold- and warm-acclimated carp were further evidenced by its thermal stability. The inactivation rate constant of myosin Ca²⁺-ATPase was 25·10^-4·s^-1 at 30°C and pH 7.0 for cold-acclimated carp, which was about 4 times as high as that for warm-acclimated carp. Light chain composition did not differ between both carp myosins. The differences in a primary structure of the heavy chain subunit was, however, clearly demonstrated between both myosins by peptide mapping.Abbreviations ATPase adenosine 5-triphosphatase - DTNB 5,5 dithio-bis-2-nitrobenzoic acid - DTT dithiothreitol - EGTA ethyleneglycol bis (-aminoethylether)-N,N,N,N-tetraacetic acid - K _D inactivation rate constant - SDS sodium dodecyl sulfate - SDS-PAGE SDS-polyacrylamide gel electrophoresis     

Comparison of ATPase activities and heavy chains of rabbit atrial and thyrotoxic ventricular myosin subfragment-1

Summary Subfragment-1 of rabbit atrial and thyrotoxic ventricular myosin (V₁ isomyosin) has been prepared and purified by DEAF-cellulose column chromatography. Pyrophosphate-polyacrylamide gel electrophoretic patterns and column chromatographic profile of the atrial subfragment differ from those of thyrotoxic ventricular myosin subfragment-1. On the other hand, Ca²⁺, Mg²⁺ and actin-activated ATPase activities of these subfragments are identical. Comparison of the peptide mapping by limited proteolysis in the presence of sodium dodecyl sulfate of the heavy and the light subunits of these subfragments reveals that the patterns for the heavy chain peptides of these subfragments are substantially similar but their light chain peptide patterns differ. The results suggest that the enzymatic and structural similarities that have been recognized between these isoenzymes using intact myosin hold true for the myosin subfragment-1.The differences between these subfragments are due to the differences in the light chains associated with them.Abbreviations EDTA Ethylene Diamine Tetra-acetic Acid - SDS Sodium Dodecyl Sulfate - S₁ myosin subfragment-1 - HC Heavy Chain - LC Light Chain     

Spatial relationship between SH1 and the actin binding site on myosin subfragment-1 surface

To examine the spatial relationship between SH1 thiol and actin binding site on subfragment-1 surface, we studied the interaction with actin of subfragment-1 whose SH1 was labeled with an iodoacetate derivative of biotin and covered with avidin. Subfragment-1--avidin complex bound F-actin and its Mg2+ ATPase activity was activated by actin. Considering the size and the location of biotin binding site on avidin, our results suggest that SH1 is separated from the actin binding site on subfragment-1 surface by at least 17-20 A.     

Effect of high hydrostatic pressure on chicken myosin subfragment-1

Hydrostatic pressure-induced structural changes in subfragment-1 (S1) of myosin molecule were studied. ATP-induced emission spectra of S1 were used to detect global structural change of S1 by pressure treatment. The fluorescence intensity of unpressurized S1 increased by addition of ATP. The increment of fluorescence of pressurized S1 up to 150 MPa was almost the same as control, whereas it became smaller above 200 MPa. ATP binding ability of S1 examined using 1, N⁶-ethenoadenosine 5′-diphosphate (-ADP) indicated that the binding of -ADP to S1 decreased in the range of 250–300 MPa. S1 pressurized below 250 MPa and unpressurized S1 similarly bound to F-actin, although binding of S1 pressurized above 250 MPa decreased. Electron microscopic observation revealed arrowhead structure in control acto-S1, while disordered arrowhead structure was observed in acto-S1 prepared from pressurized S1 at 300 MPa. S1 pressurized below 250 MPa retained the same actin activated ATPase activity as the control, whereas the activity decreased to 60% at 300 MPa. Pressure treated S1 was easily cleaved by tryptic digestion into three domains, i.e. 27 kDa (N-terminal), 50 and 20 kDa (C-terminal) fragments, which were the same as those in unpressurized one. It is concluded that pressure-induced global structural changes of S1 begin to occur about 150 MPa, and the local structural changes in ATPase and actin binding sites followed with elevating pressure to 250–300 MPa.     

Crosslinking of tropomyosin to myosin subfragment-1 in reconstituted rabbit skeletal thin filaments

Rabbit skeletal tropomyosin was labeled with the bifunctional photoactivatable crosslinker N-succinimidyl-6- (4'-azido-2'-nitrophenylamino)hexanoate. After irradiating the rigor complex composed of myosin subfragment-1, crosslinker-labeled tropomyosin, and F-actin, a crosslinked product was formed. This product was identified as a 1:1 adduct of tropomyosin and subfragment-1. This finding is in support of recent structural studies which suggest that tropomyosin and subfragment-1 are in close proximity to each other, and may be relevant to the mechanism of thin filament regulation.     

Thymol: a classical small-molecule compound that has a dual effect (potentiating and inhibitory) on myosin

The effect of thymol on the ATPase activity of myosin subfragment-1 (S1) and on the contractile properties of skinned skeletal muscle fibers was studied. At concentrations of 1.5-2 mM, thymol activated the S1 ATPase substantially and the actin-activated S1 ATPase modestly. At the same concentrations, the isometric force of skinned skeletal muscle fibers was modestly suppressed (11% at 2 mM). However, the kinetic parameters of contraction were suppressed more: the velocity of shortening and the rate of force redevelopment after shortening were suppressed by 43% and 31% at 2 mM, respectively. Thus, among other small-molecule inhibitors, thymol is unique in that it has opposite effects on the enzymatic activity and kinetic parameters of contraction. Thymol may serve as a potent tool for studying the mechanism of coupling between the ATPase reaction and contraction in muscle.     

Structural differences in the motor domain of temperature-associated myosin heavy chain isoforms from grass carp fast skeletal muscle

We determined coding sequences for three types of grass carp myosin subfragment-1 (S1) heavy chain by extending 5′-regions of the three known genes encoding light meromyosin isoforms (10 °C, intermediate and 30 °C types). The primary structures of these three S1 heavy chain isoforms showed 81.4%, 81.2%, and 97.8% identities between the 10 °C and intermediate types, between the 10 °C and 30 °C types, and between the intermediate and 30 °C types, respectively. Isoform-specific differences were clearly observed between the 10 °C type and the other two types in 97 amino acid residues. Furthermore, among these amino acid mutations, 51 mutations occurred at the conserved residue sites of S1 heavy chain from fish and homoiotherm. Additionally, the 10 °C type showed striking differences compared with the other two types in the two surface loops, loop 1 located near the ATP-binding pocket and loop 2, which is one of the actin-binding sites, suggesting that such structural differences possibly affect their motor functions. Interestingly, this 10 °C-type myosin heavy chain isolated from adult grass carp skeletal muscle was surprisingly similar to the embryonic fast-type myosin heavy chain from juvenile silver carp in the structure of S1 heavy chain, indicating that it may also function as embryonic fast-type myosin heavy chain in juvenile stage.     

The binding of skeletal muscle C-protein to F-actin, and its relation to the interaction of actin with myosin subfragment-1.

C-protein is a component of thick filaments of skeletal muscle myofibrils. It is bound to the assembly of myosin tails that forms the filament backbone. We report here that C-protein can also bind to F-actin, with a limiting stoichiometry of approximately one C-protein molecule per 3 to 5 actin subunits and a dissociation constant in the micromolar range at ionic strength 0·07. The binding is not significantly affected by ATP, calcium ions or temperature, or by the presence of tropomyosin on the actin, but it is weakened by increasing ionic strength. Myosin subfragment-1 (S-1) competes with C-protein for binding to actin. In the absence of ATP, S-1 displaces nearly all bound C-protein from actin, while in the presence of ATP, C-protein inhibits the actin activation of S-1 ATPase. Although there is no direct evidence that interaction of C-protein with actin is physiologically significant, the lenght of the C-protein molecule is sufficient so that it could make contact with the thin filaments in muscle while remaining attached to the thick filaments.     

Temperature acclimation of gross cardiovascular morphology in common carp (Cyprinus carpio)

Temperature is of fundamental importance in the functioning of the cardiovascular system of ectothermic fish, with cold-induced ventricular hypertrophy and increased red muscle mass being reported in a number of fish species upon cold acclimation. This study demonstrates a non-linear cold-induced ventricular hypertrophy in common carp (relative ventricular mass (RVM)=0.086±0.003%, 0.074±0.005% and 0.074±0.004% at 5, 15 and 25 °C, respectively), but a cold-induced atrophy of the lateral red muscle mass (RMM) with respect to total muscle mass (2.504±0.554%, 3.982±0.818% and 4.490±0.256% at 5, 15 and 25 °C, respectively). The ventricular hypertrophy in carp acclimated to 5 °C reflects a compensation against the increased workload of pumping viscous blood at low temperatures and the negative inotropic effects of the cold, whereas atrophy of the red muscle is likely due to disuse resulting from reduced locomotory activity in the cold.     

The influence of acclimation and experimental temperature on glutamate dehydrogenase of carp red lateral muscle (Cyprinus carpio L.)

The dependence of the reaction catalysed by carp red lateral muscle glutamate-dehydrogenase on acclimation and experimental temperature was studied. In addition to quantitative aspects of enzyme temperature compensation, the influence of temperature conditions on kinetic characteristics of the enzyme protein is reported. Results are discussed with respect to temperature capacity adaptation (acclimation).     

An enzyme-probe study of motile domains in the subfragment-2 region of myosin

The temperature-dependence of local melting within the subfragment-2 region of rabbit skeletal muscle myosin has been investigated using an enzyme-probe technique. Rate constants of fragmentation of two long subfragment-2 particles (61,000 Mr and 53,000 Mr per polypeptide chain) and a short subfragment-2 particle (34,000 Mr per polypeptide chain) by three different enzymes (alpha-chymotrypsin, trypsin and papain) have been determined over the temperature range 5 to 40 degrees C. We followed the time-course of digestion at specific sites at high (I = 0.50, pH 7.3) and low (physiological, I = 0.15, pH 7.3) ionic strengths by electrophoresis of the digestion products on sodium dodecyl sulfate-containing gels. All rate constants were corrected for the intrinsic temperature-dependence of the enzymes by comparison with model substrates. Normalized rate constant versus temperature profiles for the three enzyme-probes are similar in showing that local melting in long subfragment-2 (61,000 Mr) occurs in two distinct stages as was observed earlier for the intact myosin rod. Over the temperature range 5 to 25 degrees C a restricted region at Mr = 53,000 to 50,000 from the N terminus of the rod (the light meromyosin/heavy meromyosin junction) shows the highest susceptibility to proteolytic cleavage. At temperatures above 25 degrees C local melting was detected by all three enzymes at several specific sites within the hinge domain (Mr = 53,000 to 34,000). Activation energies for cleavage at the susceptible sites were similar for the three enzyme probes. They suggest that this region of the myosin rod has significantly lower thermal stability than the flanking light meromyosin and short subfragment-2 segments. These results, together with other physico-chemical studies, point to the hinge domain of the myosin cross-bridge as an important functional element in the mechanism of force generation in muscle.     

Molecular dynamics simulation of a myosin subfragment-1 docking with an actin filament

Myosins are typical molecular motor proteins, which convert the chemical energy of ATP into mechanical work. The fundamental mechanism of this energy conversion is still unknown. To explain the experimental results observed in molecular motors, Masuda has proposed a theory called the “Driven by Detachment (DbD)” mechanism for the working principle of myosins. Based on this theory, the energy used during the power stroke of the myosins originates from the attractive force between a detached myosin head and an actin filament, and does not directly arise from the energy of ATP. According to this theory, every step in the myosin working process may be reproduced by molecular dynamics (MD) simulations, except for the ATP hydrolysis step. Therefore, MD simulations were conducted to reproduce the docking process of a myosin subfragment-1 (S1) against an actin filament. A myosin S1 directed toward the barbed end of an actin filament was placed at three different positions by shifting it away from the filament axis. After 30 ns of MD simulations, in three cases out of ten trials on average, the myosin made a close contact with two actin monomers by changing the positions and the orientation of both the myosin and the actin as predicted in previous studies. Once the docking was achieved, the distance between the myosin and the actin showed smaller fluctuations, indicating that the docking is stable over time. If the docking was not achieved, the myosin moved randomly around the initial position or moved away from the actin filament. MD simulations thus successfully reproduced the docking of a myosin S1 with an actin filament. By extending the similar MD simulations to the other steps of the myosin working process, the validity of the DbD theory may be computationally demonstrated.     

Difference in the hydration water mobility around F-actin and myosin subfragment-1 studied by quasielastic neutron scattering

Hydration water is essential for a protein to perform its biological function properly. In this study, the dynamics of hydration water around F-actin and myosin subfragment-1 (S1), which are the partner proteins playing a major role in various cellular functions related to cell motility including muscle contraction, was characterized by incoherent quasielastic neutron scattering (QENS). The QENS measurements on the D₂O- and H₂O-solution samples of F-actin and S1 provided the spectra of hydration water, from which the translational diffusion coefficient (D_T), the residence time (τ_T), and the rotational correlation time (τ_R) were evaluated. The D_T value of the hydration water of S1 was found to be much smaller than that of the hydration water of F-actin while the τ_T values were similar between S1 and F-actin. On the other hand, the τ_R values of the hydration water of S1 was found to be larger than that of the hydration water of F-actin. It was also found that the D_T and τ_R values of the hydration water of F-actin are similar to those of bulk water. These results suggest a significant difference in mobility of the hydration water between S1 and F-actin: S1 has the typical hydration water, the mobility of which is reduced compared with that of bulk water, while F-actin has the unique hydration water, the mobility of which is close to that of bulk water rather than the typical hydration water around proteins.     

Conformational changes of actin induced by strong or weak myosin subfragment-1 binding

Movements of different areas of polypeptide chains within F-actin monomers induced by S1 or pPDM-S1 binding were studied by polarized fluorimetry. Thin filaments of ghost muscle were reconstructed by adding G-actin labeled with fluorescent probes attached alternatively to different sites of actin molecule. These sites were: Cys-374 labeled with 1,5-IAEDANS, TMRIA or 5-IAF; Lys-373 labeled with NBD-Cl; Lys-113 labeled with Alexa-488; Lys-61 labeled with FITC; Gln-41 labeled with DED and Cys-10 labeled with 1,5-IAEDANS, 5-IAF or fluorescein-maleimid. In addition, we used TRITC-, FITC-falloidin and e-ADP that were located, respectively, in filament groove and interdomain cleft. The data were analysed by model-dependent and model-independent methods (see appendixes). The orientation and mobility of fluorescent probes were significantly changed when actin and myosin interacted, depending on fluorophore location and binding site of actomyosin. Strong binding of S with actin leads to 1) a decrease in the orientation of oscillators of derivatives of falloidin (TRITC-falloidin, FITC-falloidin) and actin-bound nucleotide (e-ADP); 2) an increase in the orientation of dye oscillators located in the "front' surface of the small domain (where actin is viewed in the standard orientation with subdomains 1/2 and 3/4 oriented to the right and to the left, respectively); 3) a decrease in the angles of dye oscillators located on the "back" surface of subdomain-1. In contrast, a weak binding of S1 to actin induces the opposite effects in orientation of these probes. These data suggest that during the ATP hydrolysis cycle myosin heads induce a change in actin monomer (a tilt and twisting of its small domain). Presumably, these alterations in F-actin conformation play an important role in muscle contraction.     

Effect of temperature and temperature acclimation on the ryanodine sensitivity of the trout myocardium

The influence of acute temperature change and temperature acclimation on the sensitivity of contracture development to ryanodine were examined in the rainbow trout myocardium using two preparations: in vitro isolated ventricular strips and in situ working perfused hearts. Ryanodine effects in vitro were dependent on test temperature (8 and 18 °C), pacing frequency (0.2–1.5 Hz) and acclimation temperature (8 and 18 °C). At a pacing frequency of 0.2 Hz and a test temperature of 18 °C, ryanodine depressed isometric tension development in ventricular strips both from trout acclimated to 8 and 18 °C but the decrease was significantly greater in strips from 8 °C-acclimated trout. No ryanodine effect was observed in either acclimation group at a test temperature of 8°C. The effect of ryanodine in vitro was reduced or lost at pacing frequencies greater than 0.2 Hz and at 0.6 Hz ryanodine depressed tension development at 18 °C only in strips from 8 °C-acclimated trout. Ryanodine did not affect tension development at stimulation rates above 0.6 Hz in any test group. Likewise, ryanodine did not significantly impair cardiac performance of in situ working perfused heart preparations which operated at intrinsic beat frequencies in excess of 0.6 Hz. These results suggest that the sarcoplamic reticulum calcium release channel of the trout myocardium is expressed but is not functionally involved in beat-to-beat regulation of contractility at either (1) low temperature (8 °C), or (2) at routine physiological heart rate (>0.6 Hz). However, under conditions in which involvement of the sarcoplasmic reticulum is observed (18 °C and a heart rate < 0.6 Hz), prior acclimation to low temperature results in either a greater capacity of the sarcoplasmic reticulum to store releasable calcium or an increase in the amount of calcium that is in releasable form.Abbreviations bm body mass - E-C coupling, excitation-contraction coupling - IVS isometric ventricular strip - SR sarcoplasmic reticulum - TES N-tris[hydroxy-methyl]methyl-2-aminoethane sulfonic acid - WPH in situ working perfused heart     

PHI-1 induced enhancement of myosin phosphorylation in chicken smooth muscle

Herein, we provide evidence that in chicken smooth muscle, G-protein stimulation by a Rho-kinase pathway leads to an increase in myosin light chain phosphorylation. Additionally, G-protein stimulation did not increase MYPT1 phosphorylation at Thr695 or Thr850, and CPI-17, was not expressed in chicken smooth muscle. However, PHI-1 was present in chicken smooth muscle tissues. Both agonist and GTP(gamma)S stimulation result in an increase in PHI-1 phosphorylation, which is inhibited by inhibitors to both Rho-kinase (Y-27632) and (PKC) GF109203x. These data suggest that PHI-1 may act as a CPI-17 analog in chicken smooth muscle and inhibit myosin phosphatase activity during G-protein stimulation to produce Ca2+ sensitization.     

Characterisation of red and white muscle myosin heavy chain gene coding sequences from antarctic and tropical fish

To understand molecular adaptation for locomotion at different environmental temperatures, we have studied the myosin heavy chain genes as these encode the molecular motors involved. For this purpose, cDNA libraries from white (fast) and red (slow) myotomal muscle of an Antarctic and a tropical fish were constructed and from these different myosin heavy chain cDNAs were isolated. Northern and in situ hybridisation confirmed in which type of muscle these isoform genes are expressed. The cDNAs were sequenced and the structure of the ATPase sites compared. There was a marked similarity between the tropical fast myosin and the Antarctic slow myosin in the loop 1 region, which has similar amino acid side chains, charge distribution and conformation. These findings help to explain why the myofibrils isolated from white muscle of tropical fish show a lower specific ATPase activity than the white muscle of Antarctic fish but a similar activity to the Antarctic red (slow) muscle. It also provides insight into the way molecular motors in Antarctic fish have evolved to produce more power and thus ensure effective swimming at near zero temperatures by the substitution or addition of a few residues in strategic regions, which include the ATPase site.     

Effects of acclimation and egg-incubation temperature on selected temperature by hatchling western painted turtles (Chrysemys picta bellii)

The ability of hatchling turtles to detect environmental temperature differences and to effectively select preferred temperature is a function that critically impacts survival. In some turtle species, temperature preference may be influenced by embryonic and post-hatching conditions, such as egg-incubation and acclimation temperature. We tested for effects of embryonic incubation temperature (27.5 °C, 30 °C) and acclimation temperature (20 °C, 25 °C) on the selected temperature and movement patterns of 32 Chrysemys picta bellii (Reptilia: Emydidae) hatchlings in an aquatic thermal gradient of 14-34 °C and in single-temperature (20 °C, 25 °C) control tests. Among 10-11 month old hatchlings, acclimation temperature and egg-incubation temperature influenced temperature selection and movement patterns. Acclimation temperature affected activity and movement: in thermal gradient and single-temperature control tests, 25 °C-acclimated turtles relocated between chambers significantly more frequently than individuals acclimated to 20 °C. Acclimation temperature also affected temperature selection: 20 °C-acclimated turtles selected a specific temperature during gradient tests, but 25 °C-acclimated turtles did not. Among 20 °C-acclimated turtles, egg-incubation temperature was inversely related to selected temperature: hatchling turtles incubated at 27.5 °C selected the warmest temperature available (34 °C); individuals incubated at 30 °C selected the coldest temperature (14 °C). These results suggest that interactions of environmental conditions may influence post-hatching thermoregulatory behavior in C. picta bellii, a factor that ultimately affects fitness.     

Myosin light chain kinase stimulates smooth muscle myosin ATPase activity by binding to the myosin heads without phosphorylating the myosin light chain

Myosin light chain kinase (MLCK) is a multifunctional regulatory protein of smooth muscle contraction [IUBMB Life 51 (2001) 337, for review]. The well-established mode for its regulation is to phosphorylate the 20 kDa myosin light chain (MLC 20) to activate myosin ATPase activity. MLCK exhibits myosin-binding activity in addition to this kinase activity. The myosin-binding activity also stimulates myosin ATPase activity without phosphorylating MLC 20 [Proc. Natl. Acad. Sci. USA 96 (1999) 6666]. We engineered an MLCK fragment containing the myosin-binding domain but devoid of a catalytic domain to explore how myosin is stimulated by this non-kinase pathway. The recombinant fragment thus obtained stimulated myosin ATPase activity by V(max)=5.53+/-0.63-fold with K(m)=4.22+/-0.58 microM (n=4). Similar stimulation figures were obtained by measuring the ATPase activity of HMM and S1. Binding of the fragment to both HMM and S1 was also verified, indicating that the fragment exerts stimulation through the myosin heads. Since S1 is in an active form regardless of the phosphorylated state of MLC 20, we conclude that the non-kinase stimulation is independent of the phosphorylating mode for activation of myosin.