RESUMEN
We have used polyethylene glycol (PEG) to perturb the actomyosin interaction in active skinned muscle fibers. PEG is known to potentiate protein-protein interactions, including the binding of myosin to actin. The addition of 5% w/v PEG (MW 300 or 4000) to active fibers increased fiber tension and decreased shortening velocity and ATPase activity, all by 25-40%. Variation in [ADP] or [ATP] showed that the addition of PEG had little effect on the dissociation of the cross-bridge at the end of the power stroke. Myosin complexed with ADP and the phosphate analog V(i) or AlF(4) binds weakly to actin and is an analog of a pre-power-stroke state. PEG substantially enhances binding of these states both in active fibers and in solution. Titration of force with increasing [P(i)] showed that PEG increased the free energy available to drive the power stroke by about the same amount as it increased the free energy available from the formation of the actomyosin bond. Thus PEG potentiates the binding of myosin to actin in active fibers, and it provides a method for enhancing populations of some states for structural or mechanical studies, particularly those of the normally weakly bound transient states that precede the power stroke.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Fibras Musculares Esqueléticas/efectos de los fármacos , Polietilenglicoles/farmacología , Músculos Psoas/efectos de los fármacos , Actinas/química , Actomiosina/química , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/química , Compuestos de Aluminio/farmacología , Animales , Fenómenos Biomecánicos , Fluoruros/farmacología , Contracción Isométrica/efectos de los fármacos , Cinética , Miosinas/química , Unión Proteica , ConejosRESUMEN
The Mg-ATPase activity of skeletal muscle myosin subfragment 1 (S1) is reversibly eliminated when it is aggregated by the force of osmotic pressure dehydration using polyethylene glycol (PEG). Several experiments indicate nucleotides bind aggregated S1, but the effects of binding are attenuated. Compared with S1 in solution, epsilonADP binds aggregated S1 with reduced affinity, and the bound epsilonADP fluorescence intensity is more effectively quenched by acrylamide. When ATP binds aggregated S1, the tryptophan intensity increases to only 50% of the solution level. Chemical cross-linking of cys-707 to cys-697 by p-phenylenedimaleimide is less efficient for aggregated S1 x MgADP. The data are consistent with aggregated S1 being able to bind nucleotide but not being able to complete the usual conformation change(s) in response to binding. If S1 is kept from aggregating by increasing the ionic strength at the same osmotic pressure, its Mg-ATPase activity and ATP-induced tryptophan fluorescence intensity increase are normal. The combined data are consistent with an ATP hydrolysis mechanism in which S1 segmental motion is coupled to its enzymatic activity. In this model, segmental motion is mechanically constrained by aggregation; the constrained S1 can bind ATP, but it cannot complete the hydrolysis mechanism.
Asunto(s)
ATPasa de Ca(2+) y Mg(2+)/metabolismo , Músculo Esquelético/fisiología , Subfragmentos de Miosina/fisiología , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/metabolismo , Adenosina Trifosfatasas/metabolismo , Adsorción , Animales , ATPasa de Ca(2+) y Mg(2+)/antagonistas & inhibidores , ATPasas Transportadoras de Calcio/metabolismo , Proteínas de Transporte de Catión , Desecación , Durapatita/farmacología , Colorantes Fluorescentes , Cinética , Subfragmentos de Miosina/antagonistas & inhibidores , Concentración Osmolar , Presión Osmótica , Fosfatos/metabolismo , Polietilenglicoles/farmacología , ConejosRESUMEN
Variation in the concentration of orthophosphate (Pi) in actively contracting, chemically skinned muscle fibers has proved to be a useful probe of actomyosin interaction. Previous studies have shown that isometric tension (Po) decreases linearly in the logarithm of [Pi] for [Pi] > or = 200 microM. This result can be explained in terms of cross-bridge models in which the release of Pi is involved in the transition from a weakly bound, low-force actin x myosin x ADP x Pi state to a strongly bound, high-force, actin x myosin x ADP state. The 200 microM minimum [Pi] examined results from an inability to buffer the intrafiber, diffusive buildup of Pi resulting from the fiber ATPase. In the present study, we overcome this limitation by employing the enzyme purine nucleoside phosphorylase with substrate 7-methylguanosine to reduce the calculated internal [Pi] in contracting rabbit psoas fibers to < 5 microM. At 10 degrees C we find that Po continues to increase as the [Pi] decreases for [Pi] > or = 100 microM. Below this [Pi], Po is approximately constant. These results indicate that the free energy drop in the cross-bridge powerstroke is approximately 9 kT. This value is shown to be consistent with observations of muscle efficiency at physiological temperatures.
Asunto(s)
Actomiosina/metabolismo , Contracción Muscular/fisiología , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/fisiología , Fosfatos/metabolismo , Actinas/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Técnicas In Vitro , Cinética , Modelos Químicos , Miosinas/metabolismo , Pentosiltransferasa/metabolismo , ConejosRESUMEN
A series of ATP analogs, in which moieties of various sizes have been added to the gamma-phosphorus of ATP, bind to the active site of myosin and to the actomyosin complex in myofibrils and in chemically skinned fibers. The affinity of the analogs for the active site shows only a slight dependence on the size of the added moiety. Addition of even our smallest group (CH3) reduced the binding affinity of ATPgamma-CH3 for S1 to 40 microM, a factor of 10(5) less than observed for ATP. Computer molecular docking of ATP-gammaCH3 into the myosin-ADP.BeF3 crystal structure of Dictyostelium discoideum indicates no steric interference to prevent binding. This suggests that the maintenance of charge at the gamma-phosphate is crucial for tight nucleotide binding. Addition of larger groups, (1) an EPR probe (ATP-gammaSL) or (2) ADP (i.e., P1, P5-diadenosine pentaphosphate, AP5A), reduced the affinity by only approximately a factor of 10 over that of ATP-gammaCH3. In the crystal structure of S1 complexed with nucleotides, the phosphates are buried within a protein structure called "the phosphate tube". Both the bulk of the modifying groups and the lack of dependence on the size of the group are incompatible with threading of the phosphates down the Pi-tube, showing that the tube must open. Similar domain movements have been found in other proteins including members of the G-protein superfamily, a family that has structural homologies to myosin.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Miosinas/metabolismo , Animales , Unión Competitiva , Reactivos de Enlaces Cruzados , Colorantes Fluorescentes/metabolismo , Fluorometría , Cinética , Maleimidas/farmacología , Modelos Moleculares , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Unión Proteica/genética , Conejos , Especificidad por Sustrato , Compuestos de Sulfhidrilo , TriptófanoRESUMEN
Steady state measurements of the ATP turnover rate of myosin crossbridges in relaxed living mammalian muscle or in in vitro systems are complicated by other more rapid ATPase activities. To surmount these problems we have developed a technique to measure the nucleotide turnover rate of fully relaxed myosin heads in myofibrils using a fluorescent analogue of ATP (mant-ATP). Rabbit myofibrils, relaxed in 1.6 mM ATP, were rapidly mixed with an equal volume of solution containing 80 microM mant-ATP and injected into a fluorimeter. As bound ADP is released, a fraction of the myosin active sites bind mant-ATP and fluorescence emission rises exponentially, defining a rate of nucleotide turnover of 0.03 +/- 0.001 s-1 at 25 degrees C (n = 17). This rate was approximately equal to one half that of purified myosin. The turnover rates for myosin and myofibrils increased between 5 degrees and 42 degrees C, reaching 0.16 +/- 0.04 s-1 and 0.06 +/- 0.005 s-1, respectively, at 39 degrees C, the body temperature of the rabbit. If the rate observed for purified myosin occurred in vivo, it would generate more heat than is observed for resting living muscle. When myosin is incorporated into the myofilament lattice, its ATPase activity is inhibited, providing at least a partial explanation for the low rate of heat production by living resting muscle.
Asunto(s)
Adenosina Trifosfato/metabolismo , Relajación Muscular/fisiología , Músculo Esquelético/metabolismo , Miofibrillas/metabolismo , Animales , Temperatura Corporal/fisiología , Metabolismo Energético/fisiología , Colorantes Fluorescentes , Homeostasis/fisiología , Músculo Esquelético/ultraestructura , Nucleótidos/metabolismo , ConejosRESUMEN
1. Inhibition of actomyosin function by decreased pH has been proposed to account for much of the depression of muscle function during fatigue. The clearest support for this hypothesis has been from studies of skinned skeletal muscle fibre mechanics at low temperatures (< or = 15 degrees C). 2. We re-examined the effect of decreased pH (7.0-6.2) on skinned mammalian skeletal fibre mechanics at low (10 degrees C) and high (30 degrees C) temperatures, using recently developed protocols that allow reproducible mechanical data to be obtained at higher temperatures. 3. At 10 degrees C we duplicated previous observations of a significant inhibition of maximum shortening velocity (Vmax) and isometric tension (Po) by acidosis. In contrast, at the higher temperature, we found only a very minimal effect of acidosis on Vmax and a threefold reduction in the decrease in Po. 4. Thus at temperatures only slightly below physiological for mammalian skeletal muscle systems, pH plays a much less important role in the process of muscle fatigue at the cross-bridge level than has been suggested by data obtained at physiologically unrealistic temperatures.
Asunto(s)
Fatiga Muscular/fisiología , Músculos Psoas/fisiología , Acidosis/fisiopatología , Animales , Calor , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Contracción Isométrica/fisiología , Contracción Isotónica/fisiología , Contracción Muscular/fisiología , Músculos Psoas/citología , ConejosRESUMEN
Changes in the conformation of the active site of myosin subfragment-1 (S1) may be linked to the production of force during the powerstroke. We probed the conformation of the nucleotide pocket by measuring the solvent accessibility of bound mant-nucleotides. Solvent accessibility was determined by measuring the quenching of fluorescence produced by the solvent phase quencher acrylamide. The fluorescent mant moiety is attached to the ribose and is located near the outside of the pocket where it is likely to be sensitive to opening of the pocket. MantADP was highly protected from the quencher when bound to the active site of S1. A similar degree of protection was also observed for mantATP during steady-state hydrolysis by S1, and for mantADP bound to acto-S1 or to myosin in myofibrils. Assuming that S1-mantATP and actoS1-mantADP represent states at the beginning and the end of the powerstroke, respectively, we conclude that the myosin nucleotide pocket does not undergo a large conformational change during the powerstroke. However, the high degree of protection seen for mant-nucleotides is not easily explained by the open structure of the nucleotide pocket in the S1-nucleotide complex observed by x-ray diffraction.
Asunto(s)
Subfragmentos de Miosina/química , Acrilamida , Acrilamidas , Adenosina Difosfato/análogos & derivados , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión , Fenómenos Biofísicos , Biofisica , Colorantes Fluorescentes , Hidrólisis , Técnicas In Vitro , Modelos Biológicos , Contracción Muscular/fisiología , Subfragmentos de Miosina/fisiología , Conformación Proteica , Conejos , Espectrometría de Fluorescencia , ortoaminobenzoatosRESUMEN
The conformation of the active ATPase site of myosin subfragment 1 (S1) and actomyosin in myofibrils was probed by measuring the solvent accessibility of the bound ethenonucleotides epsilon ADP and epsilon ATP (during steady-state hydrolysis). Solvent accessibility was determined by measuring the quenching of fluorescence produced by the solvent-phase quencher acrylamide, 25-400 mM. The fraction of the nucleotides that were specifically bound to the active site was determined following sedimentation in the presence and absence of 5 mM ADP. In agreement with previous investigations, both epsilon ATP and epsilon ADP were almost completely protected from the quencher when bound to the active site of myosin. The solvent accessibility of both epsilon ADP and epsilon ATP varied with both temperature and ionic strength. The nucleotides became more accessible at higher temperatures and higher ionic strength. At 1 M KCl the quenching curve was biphasic, indicating that the nucleotide pocket of myosin can exist in both a closed form that allows little quenching and a more open form that allows considerable quenching. However, the transition between forms was not strongly coupled to the state of the nucleotide, with a similar protection observed for both epsilon ADP and for epsilon ATP during steady-state cycling. epsilon ADP bound to acto-S1 or to actomyosin in myofibrils displayed the same degree of protection as seen with S1 alone. A similar result is obtained during steady-state hydrolysis. Thus nucleotides in the myosin pocket do not become more accessible to the solvent when myosin binds to actin in either rigor-ADP or active complexes.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Adenosina Difosfato/análogos & derivados , Etenoadenosina Trifosfato/metabolismo , Miosinas/metabolismo , Acrilamida , Acrilamidas , Actomiosina/metabolismo , Adenosina Difosfato/metabolismo , Sitios de Unión , Colorantes Fluorescentes/metabolismo , Modelos Químicos , Subfragmentos de Miosina/metabolismo , Unión Proteica , Conformación Proteica , SolventesRESUMEN
We have investigated the ability of the nucleotides GTP, CTP, and 1-N6-etheno-2-aza-ATP (aza-ATP) to support contraction of chemically skinned rabbit psoas fibers. Working at 10 degrees C, millimolar concentrations of all nucleotides relaxed fibers in the absence of calcium. In active fibers, GTP served as a very poor substrate with isometric tension, isometric GTPase rate, and maximum shortening velocity (Vmax) all less than 10% of those obtained with ATP. Aza-ATP was only a slightly better substrate. CTP, on the other hand, was an effective substrate with mechanical parameters which were 65-100% those obtained with ATP, and with a hydrolysis rate that exceeded that of ATP. For all three ligands, Vmax followed Michaelis-Menten saturation behavior with values for Km which were from 2.5 to 12 times greater than that for ATP, showing that the analogs bound slowly to myosin in the fibers. Increasing concentrations of orthophosphate inhibited tension with CTP, to a lesser extent with aza-ATP, but not all with GTP. A combination of the mechanical data obtained in fibers with the kinetic data obtained in solution (White, H.D., Belknap, B., and Jiang, W. (1993) J. Biol. Chem. 268, 10039-10045) is used to better define the actomyosin interaction in fibers.
Asunto(s)
Contracción Isométrica/efectos de los fármacos , Músculos/fisiología , Miosinas/metabolismo , Ribonucleótidos/farmacología , Adenosina Trifosfato/farmacología , Animales , Citidina Trifosfato/farmacología , Etenoadenosina Trifosfato/análogos & derivados , Etenoadenosina Trifosfato/farmacología , Guanosina Trifosfato/farmacología , Técnicas In Vitro , Cinética , Estructura Molecular , Músculos/efectos de los fármacos , Fosfatos/farmacología , ConejosRESUMEN
We have measured the isometric tension and force-velocity relationships of glycerinated rabbit slow-twitch semimembranosus muscle as a function of MgATP concentration ([MgATP]) and have compared the results with those obtained previously from fast-twitch psoas muscle. We find that isometric tension decreases as [MgATP] increases. The magnitude of the decrease is not as great as observed in psoas. Maximum shortening velocity (Vmax) exhibits classical Michaelian saturation behavior with respect to [MgATP] with a Michaelis constant (Km) for half-maximal velocity of 18 microM and a value at saturating [MgATP] of 0.6 muscle lengths/s. Similar values were observed in fibers from soleus, another slow-twitch muscle. The corresponding values in rabbit psoas muscle are 150 microM and 1.6 lengths/s. Compared with psoas, in semimembranosus muscle Km decreases by a factor of approximately 10, whereas Vmax decreases by about a factor of 3. Thus, although in a nonphysiological regime, at low [MgATP], a "fast" muscle actually has a lower shortening velocity than a "slow" muscle.
Asunto(s)
Adenosina Trifosfato/metabolismo , Glicerol/farmacología , Contracción Muscular , Músculos/metabolismo , Animales , Contracción Isométrica , Cinética , Concentración Osmolar , Conejos , Análisis de RegresiónRESUMEN
We have investigated the ability of the photoaffinity, nonnucleotide ATP analogues, 2-[(4-azido-2-nitrophenyl) amino] ethyl triphosphate (NANTP) and 2-[(4-azido-2-nitrophenyl) amino] propyl triphosphate (PrNANTP), to support active contraction in glycerinated rabbit psoas fibers. At millimolar concentrations, in the absence of calcium, both analogues relaxed fibers. In the presence of calcium, MgNANTP produced isometric tension and stiffness that were one-half to two-thirds the values obtained in MgATP. Maximum shortening velocity and the calcium-activated, myofibrillar catalyzed rate of hydrolysis were approximately the same for MgNANTP as for MgATP. With MgNANTP as the substrate, increasing concentrations of the diphosphate analogue, MgNANDP, inhibited shortening velocity but did not change isometric tension. The addition of increased concentrations of orthophosphate (P) decreased tension while shortening velocity increased. Thus, the effects of the hydrolysis products of NANTP were quite similar to those observed previously for ADP and P in the presence of MgATP. Taken together, these observations show that MgNANTP binds to, and functions in the active site of myosin in a manner quite analogous to MgATP. Thus, the aryl azido group should serve as a valid photoaffinity label for the purine portion of the active site. In contrast, MgPrNANTP, which differs from MgNANTP only in an extra CH2 spacer between the nitrophenyl ring and the triphosphate moiety did not support isometric tension or active shortening in the presence of calcium. Fiber stiffness increased in the presence of calcium and MgPrNANTP, with a calcium-activated, myofibrillar MgPrNANTPase which was about half that obtained with MgATP. Thus, in the presence of MgPrNANTP, cross-bridges appeared to be cycling through states that were attached to actin, but not producing force.