RESUMEN
BACKGROUND: Avian influenza viruses (AIVs) are found worldwide in numerous bird species, causing significant disease in gallinaceous poultry and occasionally other species. Surveillance of wild bird reservoirs provides an opportunity to add to the understanding of the epidemiology of AIVs. METHODS: This study examined key findings from the National Avian Influenza Wild Bird Surveillance Program over a 5-year period (July 2007-June 2012), the main source of information on AIVs circulating in Australia. RESULTS: The overall proportion of birds that tested positive for influenza A via PCR was 1.9 ± 0.1%, with evidence of widespread exposure of Australian wild birds to most low pathogenic avian influenza (LPAI) subtypes (H1-13, H16). LPAI H5 subtypes were found to be dominant and widespread during this 5-year period. CONCLUSION: Given Australia's isolation, both geographically and ecologically, it is important for Australia not to assume that the epidemiology of AIV from other geographic regions applies here. Despite all previous highly pathogenic avian influenza outbreaks in Australian poultry being attributed to H7 subtypes, widespread detection of H5 subtypes in wild birds may represent an ongoing risk to the Australian poultry industry.
Asunto(s)
Gripe Aviar/epidemiología , Gripe Aviar/virología , Animales , Animales Salvajes/sangre , Animales Salvajes/virología , Anticuerpos Antivirales , Australia/epidemiología , Aves , Heces/virología , Geografía , Virus de la Influenza A/aislamiento & purificación , Gripe Aviar/sangre , Modelos Lineales , Orofaringe/virología , Reacción en Cadena de la Polimerasa , Vigilancia de la PoblaciónRESUMEN
Effects of Pi (inorganic phosphate) are relevant to the in vivo function of muscle because Pi is one of the products of ATP hydrolysis by actomyosin and by the sarcoplasmic reticulum Ca(2+) pump. We have measured the Pi sensitivity of force produced by permeabilized muscle fibres from dogfish (Scyliorhinus canicula) and rabbit. The activation conditions for dogfish fibres were crucial: fibres activated from the relaxed state at 5, 12, and 20 degrees C were sensitive to Pi, whereas fibres activated from rigor at 12 degrees C were insensitive to Pi in the range 5-25 mmol l(-1). Rabbit fibres activated from rigor were sensitive to Pi. Pi sensitivity of force produced by dogfish fibres activated from the relaxed state was greater below normal body temperature (12 degrees C for dogfish) in agreement with what is known for other species. The force-temperature relationship for dogfish fibres (intact and permeabilized fibres activated from relaxed) showed that at 12 degrees C, normal body temperature, the force was near to its maximum value.
Asunto(s)
Temperatura Corporal/fisiología , Calcio/metabolismo , Contracción Muscular/fisiología , Fibras Musculares de Contracción Rápida/metabolismo , Fosfatos/farmacología , Retículo Sarcoplasmático/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Cazón , ConejosRESUMEN
The interference fine structure of the M3 reflection on the low-angle x-ray diffraction patterns of muscle fibres is used for the measurements of axial movements of myosin heads with a precision of 0.1-0.2 nm. We have measured changes in the M3 interference profile during tension rise induced by a 5 to 30 degrees C temperature jump in thin bundles of contracting fibers from rabbit skeletal muscle. Interpreting the data with a point diffractor model gives an estimate for the axial movement of the myosin heads during force rise of less than 0.6 nm. Modifications of the point diffractor model are discussed. We show that our experimental data can be explained by a model where myosin heads bind actin in a number of structurally different states.
Asunto(s)
Modelos Moleculares , Contracción Muscular , Músculo Esquelético/química , Miosinas/química , Animales , Conejos , Difracción de Rayos XRESUMEN
Investigations were conducted into the biochemical and mechanical states of cross-bridges during isometric muscle contraction. Rapid length steps (3 or 6 nm hs(-1)) were applied to rabbit psoas fibers, permeabilized and isometric, at either 12 degrees C or 20 degrees C. Fibers were activated by photolysis of P(3)-1-(2-nitrophenyl)-ethyl ester of ATP infused into rigor fibers at saturating Ca(2+). Sarcomere length, tension, and phosphate release were recorded-the latter using the MDCC-PBP fluorescent probe. A reduction in strain, induced by a rapid release step, produced a short-lived acceleration of phosphate release. Rates of the phosphate transient and that of phases 3 and 4 of tension recovery were unaffected by step size but were elevated at higher temperatures. In contrast the amplitude of the phosphate transient was smaller at 20 degrees C than 12 degrees C. The presence of 0.5 or 1.0 mM added ADP during a release step reduced both the rate of tension recovery and the poststep isometric tension. A kinetic scheme is presented to simulate the observed data and to precisely determine the rate constants for the elementary steps of the ATPase cycle.
Asunto(s)
Actinas/metabolismo , Contracción Isométrica/fisiología , Modelos Biológicos , Proteínas Motoras Moleculares/fisiología , Fibras Musculares Esqueléticas/fisiología , Miosinas/metabolismo , Sarcómeros/fisiología , Animales , Células Cultivadas , Simulación por Computador , Elasticidad , Cinética , Conejos , Estrés MecánicoRESUMEN
Chemomechanical transduction was studied in single fibers isolated from human skeletal muscle containing different myosin isoforms. Permeabilized fibers were activated by laser-pulse photolytic release of 1.5 mM ATP from p(3)-1-(2-nitrophenyl)ethylester of ATP. The ATP hydrolysis rate in the muscle fibers was determined with a fluorescently labeled phosphate-binding protein. The effects of varying load and shortening velocity during contraction were investigated. The myosin isoform composition was determined in each fiber by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. At 12 degrees C large variations (three- to fourfold) were found between slow and fast (2A and 2A-2B) fibers in their maximum shortening velocity, peak power output, velocity at which peak power is produced, isometric ATPase activity, and tension cost. Isometric tension was similar in all fiber groups. The ATP consumption rate increased during shortening in proportion to shortening velocity. At 12 degrees C the maximum efficiency was similar (0.21-0.27) for all fiber types and was reached at a higher speed of shortening for the faster fibers. In all fibers, peak efficiency increased to approximately 0.4 when the temperature was raised from 12 degrees C to 20 degrees C. The results were simulated with a kinetic scheme describing the ATPase cycle, in which the rate constant controlling ADP release is sensitive to the load on the muscle. The main difference between slow and fast fibers was reproduced by increasing the rate constant for the hydrolysis step, which was rate limiting at low loads. Simulation of the effect of increasing temperature required an increase in the force per cross-bridge and an acceleration of the rate constants in the reaction pathway.
Asunto(s)
Actinas/metabolismo , Adenosina Trifosfato/metabolismo , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/fisiología , Miosinas/metabolismo , Adulto , Permeabilidad de la Membrana Celular , Humanos , Técnicas In Vitro , Cinética , Masculino , Persona de Mediana Edad , Isoformas de Proteínas/metabolismo , TermodinámicaRESUMEN
Myosin motors drive muscle contraction, cytokinesis and cell locomotion, and members of the myosin superfamily have been implicated in an increasingly diverse range of cell functions. Myosin can displace a bound actin filament several nanometers in a single interaction. Crystallographic studies suggest that this 'working stroke' involves bending of the myosin head between its light chain and catalytic domains. Here we used X-ray fiber diffraction to test the crystallographic model and measure the interdomain bending during force generation in an intact single muscle fiber. The observed bending has two components: an elastic distortion and an active rotation that generates force. The average bend of the force-generating myosin heads in a muscle fiber is intermediate between those in crystal structures with different bound nucleotides, and the C-terminus of the head is displaced by 7 nm along the actin filament axis compared with the in vitro conformation seen in the absence of nucleotide.
Asunto(s)
Contracción Isométrica , Proteínas Motoras Moleculares/metabolismo , Músculo Esquelético/química , Músculo Esquelético/fisiología , Miosinas/química , Miosinas/metabolismo , Actinas/metabolismo , Animales , Sitios de Unión , Biopolímeros/química , Biopolímeros/metabolismo , Dominio Catalítico , Elasticidad , Estimulación Eléctrica , Cinética , Modelos Biológicos , Modelos Moleculares , Proteínas Motoras Moleculares/química , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/citología , Nucleótidos/metabolismo , Conformación Proteica , Rana temporaria , Rotación , Relación Estructura-Actividad , Difracción de Rayos XRESUMEN
Single-molecule and macroscopic reactions of fluorescent nucleotides with myosin have been compared. The single-molecule studies serve as paradigms for enzyme-catalyzed reactions and ligand-receptor interactions analyzed as individual stochastic processes. Fluorescent nucleotides, called Cy3-EDA-ATP and Cy5-EDA-ATP, were derived by coupling the dyes Cy3.29.OH and Cy5.29.OH (compounds XI and XIV, respectively, in, Bioconjug. Chem. 4:105-111)) with 2'(3')-O-[N-(2-aminoethyl)carbamoyl]ATP (EDA-ATP). The ATP(ADP) analogs were separated into their respective 2'- and 3'-O-isomers, the interconversion rate of which was 30[OH(-)] s(-1) (0.016 h(-1) at pH 7.1) at 22 degrees C. Macroscopic studies showed that 2'(3')-O-substituted nucleotides had properties similar to those of ATP and ADP in their interactions with myosin, actomyosin, and muscle fibers, although the ATP analogs did not relax muscle as well as ATP did. Significant differences in the fluorescence intensity of Cy3-nucleotide 2'- and 3'-O-isomers in free solution and when they interacted with myosin were evident. Single-molecule studies using total internal reflection fluorescence microscopy showed that reciprocal mean lifetimes of the nucleotide analogs interacting with myosin filaments were one- to severalfold greater than predicted from macroscopic data. Kinetic and equilibrium data of nucleotide-(acto)myosin interactions derived from single-molecule microscopy now have a biochemical and physiological framework. This is important for single-molecule mechanical studies of motor proteins.
Asunto(s)
Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Músculo Esquelético/fisiología , Miosinas/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Animales , Colorantes Fluorescentes , Cinética , Contracción Muscular , Fibras Musculares Esqueléticas/fisiología , Subfragmentos de Miosina/metabolismo , Conejos , Procesos Estocásticos , Especificidad por SustratoRESUMEN
1. Structural changes following the photolytic release of ATP were observed in single, permeabilised fibres of frog skeletal muscle at 5-6 C, using time-resolved, low-angle X-ray diffraction. The structural order in the fibres and their isometric function were preserved by cross-linking 10-20 % of the myosin cross-bridges to the thin filaments. 2. The time courses of the change in force, stiffness and in intensity of the main equatorial reflections (1,0) and (1,1), of the third myosin layer line (M3) at a reciprocal spacing of (14.5 nm)-1 on the meridian and of the first myosin-actin layer line (LL1) were measured with 1 ms time resolution. 3. In the absence of Ca2+, photolytic release of ATP in muscle fibres initially in the rigor state caused the force and stiffness to decrease monotonically towards their values in relaxed muscle fibres. 4. In the presence of Ca2+, photolytic release of ATP resulted in an initial rapid decrease in force, followed by a slower increase to the isometric plateau. Muscle fibre stiffness decreased rapidly to approximately 65 % of its value in rigor. 5. In the absence of Ca2+, changes on the equator, in LL1 and in M3 occurred with a time scale comparable to that of the changes in tension and stiffness. 6. In the presence of Ca2+, the changes on the equator and LL1 occurred simultaneously with the early phase of tension decrease. The changes in the intensity of M3 (IM3) occurred on the time scale of the subsequent increase in force. The time courses of the changes in tension and IM3 were similar following the photolytic release of 0. 33 or 1.1 mM ATP. However the gradual return towards the rigor state began earlier when only 0.33 mM ATP was released. 7. In the presence of Ca2+, the time course of changes in IM3 closely mimicked that of force development following photolytic release of ATP. This is consistent with models that propose that force development results from a change in the average orientation of cross-bridges, although other factors, such as their redistribution, may also be involved.
Asunto(s)
Adenosina Trifosfato/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Fotólisis , Animales , Calcio/metabolismo , Elasticidad , Microanálisis por Sonda Electrónica , Corazón/fisiología , Corazón/efectos de la radiación , Rayos Láser , Masculino , Contracción Muscular/fisiología , Miocardio/metabolismo , Rana temporaria , Factores de TiempoRESUMEN
Structural changes induced by Joule temperature jumps (T-jumps) in frog muscle fibers were monitored using time-resolved x-ray diffraction. Experiments made use of single, permeabilized fibers that were fully activated after slight cross-linking with 1-ethyl-3-[3-dimethylamino)propyl]carbodiimide to preserve their structural order. After T-jumps from 5-6 to approximately 17 degrees C and then on to approximately 30 degrees C, tension increased by a factor of 1.51 and 1.84, respectively, whereas fiber stiffness did not change with temperature. The tension rise was accompanied by a decrease in the intensity of the (1, 0) equatorial x-ray reflection by 15 and 26% (at approximately 17 and approximately 30 degrees C) and by an increase in the intensity of the M3 myosin reflection by 20% and 41%, respectively. The intensity of the (1,1) equatorial reflection increased slightly. The peak of the intensity on the 6th actin layer line shifted toward the meridian with temperature. The intensity of the 1st actin layer line increased from 12% (of its rigor value) at 5-6 degrees C to 36% at approximately 30 degrees C, so that the fraction of the cross-bridges labeling the actin helix estimated from this intensity increased proportionally to tension from approximately 35% at 5-6 degrees C to approximately 60% at approximately 30 degrees C. This suggests that force is generated during a transition of nonstereo-specifically attached myosin cross-bridges to a stereo-specific binding state.
Asunto(s)
Actomiosina/química , Contracción Muscular , Fibras Musculares Esqueléticas/química , Actomiosina/ultraestructura , Animales , Calcio/química , Permeabilidad de la Membrana Celular , Reactivos de Enlaces Cruzados/química , Etildimetilaminopropil Carbodiimida/análogos & derivados , Etildimetilaminopropil Carbodiimida/química , Cinética , Fibras Musculares Esqueléticas/ultraestructura , Rana temporaria , Temperatura , Difracción de Rayos XRESUMEN
1. The relationship between mechanical power output and the rate of ATP hydrolysis was investigated in segments of permeabilized fibres isolated from rabbit psoas muscle. 2. Contractions were elicited at 12 degrees C by photolytic release of ATP from the P3 -1-(2-nitrophenyl) ester of ATP (NPE-caged ATP). Inorganic phosphate (Pi) release was measured by a fluorescence method using a coumarin-labelled phosphate binding protein. Force and sarcomere length were also monitored. 3. ATPase activity was determined from the rate of appearance of Pi during each phase of contraction. The ATPase rate was 10.3 s-1 immediately following release of ATP and 5. 1 s-1 during the isometric phase prior to the applied shortening. It rose hyperbolically with shortening velocity, reaching 18.5 s-1 at a maximal shortening velocity > 1 ML s-1 (muscle lengths s-1). 4. Sarcomeres shortened at 0.09 ML s-1 immediately following the photolytic release of ATP and at 0.04 ML s-1 prior to the period of applied shortening. The high initial ATPase rate may be largely attributed to initial sarcomere shortening. 5. During shortening, maximal power output was 28 W l-1. Assuming the free energy of hydrolysis is 50 kJ mol-1, the efficiency of contraction was calculated from the power output at each shortening velocity. The maximum efficiency was 0.36 at a shortening velocity of 0.27 ML s-1, corresponding to a force level 51 % of that in the isometric state. 6. At the maximal shortening velocity, only 10 % of the myosin heads are attached to the thin filaments at any one time.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Contracción Muscular/fisiología , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/fisiología , Fosfatos/metabolismo , Sarcómeros/fisiología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Animales , Técnicas In Vitro , Cinética , Fotólisis , Conejos , TemperaturaRESUMEN
Muscle contraction is driven by a change in shape of the myosin head region that links the actin and myosin filaments. Tilting of the light-chain domain of the head with respect to its actin-bound catalytic domain is thought to be coupled to the ATPase cycle. Here, using X-ray diffraction and mechanical data from isolated muscle fibres, we characterize an elastic bending of the heads that is independent of the presence of ATP. Together, the tilting and bending motions can explain force generation in isometric muscle, when filament sliding is prevented. The elastic strain in the head is 2.0-2.7 nm under these conditions, contributing 40-50% of the compliance of the muscle sarcomere. We present an atomic model for changes in head conformation that accurately reproduces the changes in the X-ray diffraction pattern seen when rapid length changes are applied to muscle fibres both in active contraction and in the absence of ATP. The model predictions are relatively independent of which parts of the head are assumed to bend or tilt, but depend critically on the measured values of filament sliding and elastic strain.
Asunto(s)
Contracción Muscular/fisiología , Miosinas/fisiología , Actinas/química , Actinas/fisiología , Adenosina Trifosfato/fisiología , Animales , Elasticidad , Proteínas Motoras Moleculares , Fibras Musculares Esqueléticas/fisiología , Miosinas/química , Conformación Proteica , Rana temporaria , Difracción de Rayos XRESUMEN
The rate of release of inorganic phosphate (Pi) from cycling cross-bridges in rabbit portal-anterior mesenteric vein smooth muscle was determined by following the fluorescence of the Pi-reporter, MDCC-PBP (Brune, M., J. L. Hunter, S. A. Howell, S. R. Martin, T. L. Hazlett, J. E. T. Corrie, and M. R. Webb. 1998. Biochemistry. 37:10370-10380). Cross-bridge cycling was initiated by photolytic release of ATP from caged-ATP in Triton-permeabilized smooth muscles in rigor. When the regulatory myosin light chains (MLC20) had been thiophosphorylated, the rate of Pi release was biphasic with an initial rate of 80 microM s-1 and amplitude 108 microM, decreasing to 13.7 microM s-1. These rates correspond to fast and slow turnovers of 1.8 s-1 and 0.3 s-1, assuming 84% thiophosphorylation of 52 microM myosin heads. Activation by Ca2+-dependent phosphorylation subsequent to ATP release resulted in slower Pi release, paralleling the rate of contraction that was also slower than after thiophosphorylation, and was also biphasic: 51 microM s-1 and 13.2 microM s-1. These rates suggest that the activity of myosin light chain kinase and phosphatase ("pseudo-ATPase") contributes <20% of the ATP usage during cross-bridge cycling. The extracellular "ecto-nucleotidase" activity was reduced eightfold by permeabilization, conditions in which the ecto-ADPase was 17% of the ecto-ATPase. Nevertheless, the remaining ecto-ATPase activity reduced the precision of the estimate of cross-bridge ATPase. We conclude that the transition from fast to slow ATPase rates reflects the properties and forces directly acting on cross-bridges, rather than the result of a time-dependent decrease in activation (MLC20 phosphorylation) occurring in intact smooth muscle. The mechanisms of slowing may include the effect of positive strain on cross-bridges, inhibition of the cycling rate by high affinity Mg-ADP binding, and associated state hydrolysis.
Asunto(s)
Músculo Liso Vascular/metabolismo , Fosfatos/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Animales , Fenómenos Biofísicos , Biofisica , Técnicas In Vitro , Cinética , Contracción Muscular/fisiología , Músculo Liso Vascular/fisiología , Miosinas/metabolismo , Permeabilidad , Fosforilación , Fotólisis , Vena Porta/metabolismo , Vena Porta/fisiología , ConejosRESUMEN
Inorganic phosphate (Pi) release was determined by means of a fluorescent Pi-probe in single permeabilized rabbit soleus and psoas muscle fibers. Measurements of Pi release followed photoliberation of approximately 1.5 mM ATP by flash photolysis of NPE-caged ATP in the absence and presence of Ca2+ at 15 degrees C. In the absence of Ca2+, Pi release occurred with a slow rate of 11 +/- 3 microM . s-1 (n = 3) in soleus fibers and 23 +/- 1 microM . s-1 (n = 10) in psoas fibers. At saturating Ca2+ concentrations (pCa 4.5), photoliberation of ATP was followed by rapid force development. The initial rate of Pi release was 0.57 +/- 0.05 mM . s-1 in soleus (n = 13) and 4.7 +/- 0.2 mM . s-1 in psoas (n = 23), corresponding to a rate of Pi release per myosin head of 3.8 s-1 in soleus and 31.5 s-1 in psoas. Pi release declined at a rate of 0.48 s-1 in soleus and of 5.2 s-1 in psoas. Pi release in soleus was slightly faster in the presence of an ATP regenerating system but slower when 0.5 mM ADP was added. The reduction in the rate of Pi release results from an initial redistribution of cross-bridges over different states and a subsequent ADP-sensitive slowing of cross-bridge detachment.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Fibras Musculares de Contracción Rápida/metabolismo , Fibras Musculares de Contracción Lenta/metabolismo , Músculo Esquelético/fisiología , Fosfatos/metabolismo , Adenosina Difosfato/farmacología , Adenosina Trifosfato/metabolismo , Animales , Calcio/farmacología , Proteínas Portadoras/metabolismo , Cumarinas/metabolismo , Colorantes Fluorescentes , Cinética , Contracción Muscular/fisiología , Proteínas de Unión a Fosfato , Fotólisis , Músculos Psoas/fisiología , Conejos , Sarcómeros/metabolismoRESUMEN
Muscle proteins utilise the hydrolysis of ATP to provide the energy for force development and the production of mechanical work. We have developed a technique with high sensitivity and time resolution to probe as directly as possible the link between ATPase activity, force development and muscle shortening. The ATPase activity was recorded in real time during contraction and shortening of permeabilised muscle fibres of rabbit skeletal muscle by measuring fluorescence changes associated with the binding of inorganic phosphate, a product of ATPase activity, to a genetically engineered phosphate binding protein labelled with a coumarin fluorophore. The muscle shortening velocity was found to affect directly the ATPase activity, with up to a five-fold increase during shortening at moderate velocities, and a decrease in activity during slow stretch.
Asunto(s)
Adenosina Trifosfatasas/fisiología , Contracción Muscular/fisiología , Músculo Esquelético/fisiología , Adenosina Trifosfato/fisiología , Animales , Activación Enzimática , ConejosRESUMEN
Muscle force is generated by myosin crossbridges interacting with actin. As estimated from stiffness and equatorial X-ray diffraction of muscle and muscle fibres, most myosin crossbridges are attached to actin during isometric contraction, but a much smaller fraction is bound stereospecifically. To determine the fraction of crossbridges contributing to tension and the structural changes that attached crossbridges undergo when generating force, we monitored the X-ray diffraction pattern during temperature-induced tension rise in fully activated permeabilized frog muscle fibres. Temperature jumps from 5-6 degrees C to 16-19 degrees C initiated a 1.7-fold increase in tension without significantly changing fibre stiffness or the intensities of the (1,1) equatorial and (14.5 nm)(-1) meridional X-ray reflections. However, tension rise was accompanied by a 20% decrease in the intensity of the (1,0) equatorial reflection and an increase in the intensity of the first actin layer line by approximately 13% of that in rigor. Our results show that muscle force is associated with a transition of the crossbridges from a state in which they are nonspecifically attached to actin to one in which stereospecifically bound myosin crossbridges label the actin helix.
Asunto(s)
Actinas/fisiología , Músculos/fisiología , Miosinas/fisiología , Animales , Fenómenos Biomecánicos , Técnicas In Vitro , Contracción Muscular , Fibras Musculares Esqueléticas/fisiología , Rana temporaria , Temperatura , Difracción de Rayos XRESUMEN
1. The rate of appearance of inorganic phosphate (Pi) and hence the ATPase activity of rabbit psoas muscle in single permeabilized muscle fibres initially in rigor was measured following laser flash photolysis of the P3-1-(2-nitrophenyl)ethyl ester of ATP (NPE-caged ATP) in the presence and absence of Ca2+. Pi appearance was monitored from the fluorescence signal of a Pi-sensitive probe, MDCC-PBP, a coumarin-labelled A197C mutant of the phosphate-binding protein from Escherichia coli. Fibres were immersed in oil to optimize the fluorescence signal and to obviate diffusion problems. The ATPase activity was also measured under similar conditions from the rate of NADH disappearance using an NADH-linked coupled enzyme assay. 2. On photolysis of NPE-caged ATP in the presence of Ca2+ at 20 degrees C, the fluorescence increase of MDCC-PBP was non-linear with time. ATPase activity was 41 s-1 in the first turnover based on a myosin subfragment 1 concentration of 150 microM. This was calculated from a linear regression of the fluorescence signal reporting 20-150 microM of Pi release. Tension was at 67% of its isometric level by the time 150 microM Pi was released. ATPase activities were 36 and 31 s-1 for Pi released in the ranges of 150-300 microM and 300-450 microM, respectively. The ATPase activity had a Q10 value of 2.9 based on measurements at 5, 12 and 20 degrees C. 3. An NADH-linked assay showed the ATPase activity had a lower limit of 12.7 s-1 at 20 degrees C. The response to photolytic release of ADP showed that the rate of NADH disappearance was partially limited by the flux through the coupled reactions. Simulations indicated that the linked assay data were consistent with an initial ATPase activity of 40 s-1. 4. On photolysis of NPE-caged ATP in the absence of Ca2+ the ATPase activity was 0.11 s-1 at 20 degrees C with no discernible rapid transient phase of Pi release during the first turnover of the ATPase. 5. To avoid the rigor state, the ATPase rate in the presence of Ca2+ was also measured on activation from the relaxed state by photolytic release of Ca2+ from a caged Ca2+ compound, nitrophenyl-EGTA. At 5 degrees C the ATPase rate was 5.8 and 4.0 s-1 in the first and second turnovers, respectively. These rates are comparable to those when NPE-caged ATP was used. 6. The influence of ADP and Pi on the ATPase activities was measured using the MDCC-PBP and NADH-linked assays, respectively. ADP (0.5 mM) decreased the initial ATPase rate by 23%. Pi (10 mM) had no significant effect. Inhibition by ADP, formed during ATP hydrolysis, contributed to the decrease of ATPase activity with time. 7. The MDCC-PBP assay and NPE-caged ATP were used to measure the ATPase rate in single permeabilized muscle fibres of the semitendinosus muscle of the frog. At 5 degrees C in the presence of Ca2+ the ATPase activity was biphasic being 15.0 s-1 during the first turnover (based on 180 microM myosin subfragment 1). Tension was 74% of its isometric level by the time 180 microM Pi was released. During the third turnover the ATPase rate decreased to about 20% of that during the first turnover. 8. ATPase activity in isometric rabbit muscle fibres during the first few turnovers is about an order of magnitude greater than that when a steady state is reached. Possible reasons and the consequences for understanding the mechanism of muscular contraction are discussed.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Contracción Isométrica , Fibras Musculares Esqueléticas/metabolismo , Fosfatos/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Proteínas Portadoras/metabolismo , Cumarinas/metabolismo , Colorantes Fluorescentes/metabolismo , Cinética , Masculino , NAD/metabolismo , Proteínas de Unión a Fosfato , Músculos Psoas/metabolismo , Conejos , Rana temporaria , Sarcómeros/metabolismo , Tendones , Difracción de Rayos XRESUMEN
We show prolonged contraction of permeabilized muscle fibers of the frog during which structural order, as judged from low-angle x-ray diffraction, was preserved by means of partial cross-linking of the fibers using the zero-length cross-linker 1-ethyl-3-[3-dimethylamino)propyl]carbodiimide. Ten to twenty percent of the myosin cross-bridges were cross-linked, allowing the remaining 80-90% to cycle and generate force. These fibers displayed a well-preserved sarcomeric order and mechanical characteristics similar to those of intact muscle fibers. The intensity of the brightest meridional reflection at 14.5 nm, resulting from the projection of cross-bridges evenly spaced along the myofilament length, decreased by 60% as a relaxed fiber was deprived of ATP and entered the rigor state. Upon activation of a rigorized fiber by the addition of ATP, the intensity of this reflection returned to 97% of the relaxed value, suggesting that the overall orientation of cross-bridges in the active muscle was more perpendicular to the filament axis than in rigor. Following a small-amplitude length step applied to the active fibers, the reflection intensity decreased for both releases and stretches. In rigor, however, a small stretch increased the amplitude of the reflection by 35%. These findings show the close link between cross-bridge orientation and tension changes.
Asunto(s)
Contracción Muscular/fisiología , Músculo Esquelético/fisiología , Actinas/química , Animales , Fenómenos Biomecánicos , Fenómenos Biofísicos , Biofisica , Pollos , Creatina Quinasa/metabolismo , Reactivos de Enlaces Cruzados , Etildimetilaminopropil Carbodiimida , Técnicas In Vitro , Estructura Molecular , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/química , Miosinas/química , Rana temporaria , Difracción de Rayos XRESUMEN
The effects of both the P3-1-(2-nitrophenyl)ethyl ester of adenosine 5'-triphosphate (NPE-caged ATP) and its separate diastereoisomers, and the P3-3',5'-dimethoxybenzoin ester of ATP (DMB-caged ATP) were studied on the unloaded shortening velocity of glycerinated rabbit psoas muscle fibres. The unloaded shortening velocities of the active fibres were measured as a function of ATP concentration (0.1-5 mM) using the 'slack-test' with and without 2 mM caged ATP. Shortening velocity followed a Michaelis-Menten relationship with ATP concentration, the Km for ATP being 170 microM. The caged ATP compounds inhibited shortening velocity, in a manner consistent with competitive inhibition, with a Ki of 1-2 mM. The R- and S-diastereoisomers of NPE-caged ATP showed the same degree of competitive inhibition of the shortening velocity, as did DMB-caged ATP. These observations suggest that caged ATP compounds bind to the ATPase site of the actomyosin where they compete with the substrate, Mg2+ ATP.