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1.
Raman spectra, in the frequency region of the protein vibrations, of intact single muscle fibers of the giant barnacle are presented. Strong bands at 1521 and 1156 cm-1 in the spectra are attributed to resonance-enhanced Raman bands of membrane-bound beta-carotene. Many bands of the myofibrillar proteins are also observed, and at least three spectral features confirm that these proteins adopt a predominantly alpha-helical structure: (1) the amide I band at 1648 cm-1, (2) the weak scattering in the amide III region, and (3) a strong skeletal C-C stretching band at 939 cm-1. Deuterated fibers have also been examined in order to find the exact shape of the amide III band. The presence in the fibers of paramyosin, which is only found in catch muscles, is also apparent from the spectra.  相似文献   

2.
The myosin lattice spacing of single intact muscle fibers of the frog, Rana temporaria, was studied in Ringer's solution (standard osmolarity 230 mOsm) and hyper- and hypotonic salines (1.4 and 0.8 times standard osmolarity respectively) in the relaxed state, during "fixed end" tetani, and during shortening, using synchrotron radiation. At standard tonicity, a tetanus was associated with an initial brief lattice expansion (and a small amount of sarcomere shortening), followed by a slow compression (unaccompanied by sarcomere length changes). In hypertonic saline (myosin lattice compressed by 8.1%), these spacing changes were suppressed, in hypotonic saline (lattice spacing increased by 7.5%), they were enhanced. During unloaded shortening of activated fibers, a rapid lattice expansion occurred at all tonicities, but became larger as tonicity was reduced. This expansion was caused in part by the change in length of the preparation, but also by a recoil of a stressed radial compliance associated with axial force. The lattice spacing during unloaded shortening was equal to or occasionally greater than predicted for a relaxed fiber at that sarcomere length, indicating that the lattice compression associated with activation is rapidly reversed upon loss of axial force. Lattice recompression occurred upon termination of shortening under standard and hypotonic conditions, but was almost absent under hypertonic conditions. These observations indicate that axial cross-bridge tension is associated with a compressive radial force in intact muscle fibers at full overlap; however, this radial force exhibits a much greater sensitivity to lattice spacing than does the axial force.  相似文献   

3.
Summary Control of mitochondrial respiration depends on ADP availability to the F1ATPase. An electrochemical gradient of ADP and ATP across the mitochondrial inner membrane is maintained by the adenine nucleotide translocase which provides ADP to the matrix for ATP synthesis and ATP for energy-dependent processes in the cytosol. Mitochondrial respiration is responsive to the cytosolic phosphorylation potential, ATP/ADP · Pi which is in apparent equilibrium with the first two sites in the electron transport chain. Conventional measures of free adenine nucleotides is a confounding issue in determining cytosolic and mitochondrial phosphorylation potentials. The advent of phosphorus-31 nuclear magnetic resonance (P-31 NMR) allows the determination of intracellular free concentrations of ATP, creatine-P and Pi in perfused muscle in situ. In the glucose-perfused heart, there is an absence of correlation between the cytosolic phosphorylation potential as determined by P-31 NMR and cardiac oxygen consumption over a range of work loads. These data suggest that contractile work leads to increased generation of mitochondrial NADH so that ATP production keeps pace with myosin ATPase activity. The mechanism of increased ATP synthesis is referred to as stimulusre-sponse-metabolism coupling. In muscle, increased contractility is a result of interventions which increase cytosolic free Ca2+ concentrations. The Ca2- signal thus generated increases glycogen breakdown and myosin ATPase in the cytosol. This signal is concomitantly transmitted to the mitochondria which respond to small increases in matrix Ca2+ by activation of Ca2+-sensitive dehydrogenases. The Ca2+-activated dehydrogenase activities are key rate-controlling enzymes in tricarboxylic acid cycle flux, and their activation by Ca2- leads to increased pyridine nucleotide reduction and oxidative phosphorylation. These observations which have been consistent in preparations both in vitro and in situ do not obviate a role for ADP control of muscle respiration, but do explain, in part, the lack of dramatic fluctuations in the cytosolic phosphorylation potential over a large range of contractile activities.  相似文献   

4.
Laser Raman spectroscopy has been used to investigate the state of water in intact single muscle fibers of the giant barnacle (Balanus nulilus). The spectra in the region of the O-H (or O-2H) stretching modes of water in unfrozen fibers show that there is no appreciable difference between the shape and relative intensity of the Raman bands due to the water molecules located inside a muscle fiber and those of the corresponding bands in the spectrum of pure water. The presence of significant amounts of “structured” intracellular water, greater than approx. 5% of the total water content, in these fibers is thus excluded. The Raman spectra of frozen fibers have also been recorded in order to evaluate the amount of intracellular water which remains unfrozen at temperatures below the normal freezing point of water. We have been able to reproduce these spectra by assuming that the spectrum of a frozen fiber is the sum of the individual spectra of water and ice. To calculate the amount of unfrozen water from these curve fittings, it was also necessary to determine the intensities of the water and ice Raman bands relative to one another. We have found the I(ice)/I(water) ratio is 1.07 ± 0.01 for H2O and 1.05 ± 0.03 for 2H2O With these figures, we have calculated that for a fiber with a normal water content of 80%, 20% of the water molecules remain in the supercooled state at ?5°C, which corresponds to 1 g of water per of fiber dry weight. This amount of bound water was also found to be independent of the water content of the fibers.  相似文献   

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6.
Equatorial x-ray diffraction pattern intensities (I10 and I11), fiber stiffness and sarcomere length were measured in single, intact muscle fibers under isometric conditions and during constant velocity (ramp) shortening. At the velocity of unloaded shortening (Vmax) the I10 change accompanying activation was reduced to 50.8% of its isometric value, I11 reduced to 60.7%. If the roughly linear relation between numbers of attached bridges and equatorial signals in the isometric state also applies during shortening, this would predict 51-61% attachment. Stiffness (measured using 4 kHz sinusoidal length oscillations), another putative measure of bridge attachment, was 30% of its isometric value at Vmax. When small step length changes were applied to the preparation (such as used for construction of T1 curves), no equatorial intensity changes could be detected with our present time resolution (5 ms). Therefore, unlike the isometric situation, stiffness and equatorial signals obtained during ramp shortening are not in agreement. This may be a result of a changed crossbridge spatial orientation during shortening, a different average stiffness per attached crossbridge, or a higher proportion of single headed crossbridges during shortening.  相似文献   

7.
The AM loading of a number of different fluorescent Ca2+ indicators was compared in intact single fibers of frog muscle. Among the 13 indicators studied, loading rates (the average increase in the fiber concentration of indicator per first 60 min of loading) varied approximately 100-fold, from approximately 3 microM/h to >300 microM/h (16 degrees C). Loading rates were strongly dependent on the molecular weight of the AM compounds, with the rate increasing steeply as molecular weight decreased below approximately 850. Properties of delta F/F (the Ca2(+)-related fluorescence signal observed with fiber stimulation) were also measured in AM-loaded fibers and compared with those previously reported for fibers microinjected with indicator. In general, the time course of delta F/F was very similar with AM-loading and microinjection; however, the amplitude of delta F/F was usually smaller with AM-loading. There was a strong correlation between the rate of indicator loading and the value of the parameter f (the ratio of the amplitude of delta F/F in AM-loaded versus microinjected fibers). For indicators with small loading rates (<10 microM/h, N = 5), f values were generally small (< or =0.4, N = 4); whereas with large loading rates (>100 microM/h, N = 4), f values were large (> or =0.8, N = 4). This suggests that, with any AM indicator, a small concentration may associate nonspecifically with the fiber (either the indicator is incompletely de-esterified or, if completely de-esterified, not located in the myoplasmic compartment). If the loaded concentration is small, the nonspecific indicator will present a significant source of error in the estimation of [Ca2+]i.  相似文献   

8.
9.
Ca influx and efflux in single twitch muscle fibers were determined by the movement of 45Ca. The isotope was assayed by counting the center 1 cm of a fiber while it was in nonradioactive Rnger's solution. The average resting influx in 1.0 mM Ca Ringer's was 0.26 pM Ca/cm2. sec for 5 to 20 min influx periods. The average additional influx upon stimulation in 1.0 mM Ca was 0.73 pM Ca/cm2. twitch. The efflux after both resting and stimulated 45Ca influx can be described by a single exponential curve with an average time constant of 125 min. This relationship is an indication of Ca exchange with a single intracellular compartment. This compartment contains an estimated 47% of the total muscle Ca at 1.0 mM Ca. When the Ca in the Ringer was reduced to 0.5 mM Ca, both the resting and stimulated Ca fluxes decreased. When Ca was raised to 1.8 mM, the stimulated influxes increased but the resting influx did not.  相似文献   

10.
Length-dependent electromechanical coupling in single muscle fibers   总被引:2,自引:2,他引:0       下载免费PDF全文
In single muscle fibers from the giant barnacle, a small decrease in muscle length decreases both the calcium activation and the peak isometric tension produced by a constant current stimulus. The effect is most pronounced if the length change immediately precedes the stimulation. In some cases, the decrease in tension with shortening can be accounted for almost entirely by a decrease in calcium release rather than changes in mechanical factors such as filament geometry. During the constant current stimulation the muscle membrane becomes more depolarized at longer muscle lengths than at the shorter muscle lengths. Under voltage clamp conditions, when the membrane potential is kept constant during stimulation, there is little length dependence of calcium release. Thus, the effect of length on calcium release is mediated through a change in membrane properties, rather than an effect on a subsequent step in excitation-contraction coupling. Stretch causes the unstimulated fiber membrane to depolarize by about l mV while release causes the fiber membrane to hyperpolarize by about the same amount. The process causing this change in potential has an equilibrium potential nearly 10 mV hyperpolarized from the resting level. This change in resting membrane potential with length may account for the length dependence of calcium release.  相似文献   

11.
The myoplasmic free Ca2+concentration([Ca2+]i)was measured in intact single fibers from mouse skeletal muscle withthe fluorescent Ca2+ indicatorindo 1. Some fibers were perfused in a solution in which theconcentration of Na+ was reducedfrom 145.4 to 0.4 mM (low-Na+solution) in an attempt to activate reverse-modeNa+/Ca2+exchange (Ca2+ entry in exchangefor Na+ leaving the cell). Undernormal resting conditions, application oflow-Na+ solution only increased[Ca2+]iby 5.8 ± 1.8 nM from a mean resting[Ca2+]iof 42 nM. In other fibers,[Ca2+]iwas elevated by stimulating sarcoplasmic reticulum (SR)Ca2+ release with caffeine (10 mM)and by inhibiting SR Ca2+ uptakewith2,5-di(tert-butyl)-1,4-benzohydroquinone(TBQ; 0.5 µM) in an attempt to activate forward-modeNa+/Ca2+exchange (Ca2+ removal from thecell in exchange for Na+ influx).These two agents caused a large increase in[Ca2+]i,which then declined to a plateau level approximately twice the baseline[Ca2+]iover 20 min. If the cell was allowed to recover between exposures tocaffeine and TBQ in a solution in whichCa2+ had been removed, theincrease in[Ca2+]iduring the second exposure was very low, suggesting thatCa2+ had left the cell during theinitial exposure. Application of caffeine and TBQ to a preparation inlow-Na+ solution produced a large,sustained increase in[Ca2+]iof ~1 µM. However, when cells were exposed to caffeine and TBQ in alow-Na+ solution in whichCa2+ had been removed, a sustainedincrease in[Ca2+]iwas not observed, although[Ca2+]iremained higher and declined slower than in normalNa+ solution. This suggests thatforward-modeNa+/Ca2+exchange contributed to the fall of[Ca2+]iin normal Na+ solution, but whenextracellular Na+ was low, aprolonged elevation of[Ca2+]icould activate reverse-modeNa+/Ca2+exchange. The results provide evidence that skeletal muscle fibers possess aNa+/Ca2+exchange mechanism that becomes active in its forward mode when [Ca2+]iis increased to levels similar to that obtained during contraction.

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12.
K.S. Cheah  J.C. Waring 《BBA》1983,723(1):45-51
The effect of trifluoperazine on the respiration of porcine liver and skeletal muscle mitochondria was investigated by polarographic and spectroscopic techniques. Low concentrations of trifluoperazine (88 nmol/mg protein) inhibited both the ADP- and Ca2+-stimulated oxidation of succinate, and reduced the values of the respiratory control index and the ADPO and Ca2+O ratio. High concentrations inhibited both succinate and ascorbate plus tetramethyl-p-phenylenediame (TMPD) oxidations, and uncoupler (carbonyl cyanide p-trifluromethoxyphenylhydrazone) and Ca2+-stimulated respiration. Porcine liver mitochondria were more sensitive to trifluoperazine than skeletal muscle mitochondria. Trifluoperazine inhibited the electron transport of succinate oxidation of skeletal muscle mitochondria within the cytochrome b-c1 and cytochrome c1-aa3 segments of the respiratory chain system. 233 nmol trifluoperazine/mg protein inhibited the aerobic steady-state reduction of cytochrome c1 by 92% with succinate as substrate, and of cytochrome c and cytochrome aa3 by 50–60% with ascorbate plus TMPD as electron donors. Trifluoperazine can thus inhibit calmodulin-independent reactions particularly when used at high concentrations.  相似文献   

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16.
The 500 to 1,800-cm-1 region of the Raman spectra of intact single muscle fibers from the giant barnacle are dominated by bands caused by the protein component of the fibers. The frequency and the intensity of the conformationally sensitive bands indicate that the contractile proteins adopt a predominantly alpha-helical structure and are not affected when the contractile state of the fibers is changed from relaxed to contracted by addition of ATP and Ca. However, the contraction induces a decrease of the scattering intensity of some of the Raman bands caused by the acidic and tryptophan side chains, showing that these amino acids are involved during the generation of tension.  相似文献   

17.
18.
Calcium sparks were studied in frog intact skeletal muscle fibers using a home-built confocal scanner whose point-spread function was estimated to be approximately 0.21 microm in x and y and approximately 0.51 microm in z. Observations were made at 17-20 degrees C on fibers from Rana pipiens and Rana temporaria. Fibers were studied in two external solutions: normal Ringer's ([K(+)] = 2.5 mM; estimated membrane potential, -80 to -90 mV) and elevated [K(+)] Ringer's (most frequently, [K(+)] = 13 mM; estimated membrane potential, -60 to -65 mV). The frequency of sparks was 0.04-0.05 sarcomere(-1) s(-1) in normal Ringer's; the frequency increased approximately tenfold in 13 mM [K(+)] Ringer's. Spark properties in each solution were similar for the two species; they were also similar when scanned in the x and the y directions. From fits of standard functional forms to the temporal and spatial profiles of the sparks, the following mean values were estimated for the morphological parameters: rise time, approximately 4 ms; peak amplitude, approximately 1 DeltaF/F (change in fluorescence divided by resting fluorescence); decay time constant, approximately 5 ms; full duration at half maximum (FDHM), approximately 6 ms; late offset, approximately 0.01 DeltaF/F; full width at half maximum (FWHM), approximately 1.0 microm; mass (calculated as amplitude x 1.206 x FWHM(3)), 1.3-1.9 microm(3). Although the rise time is similar to that measured previously in frog cut fibers (5-6 ms; 17-23 degrees C), cut fiber sparks have a longer duration (FDHM, 9-15 ms), a wider extent (FWHM, 1.3-2.3 microm), and a strikingly larger mass (by 3-10-fold). Possible explanations for the increase in mass in cut fibers are a reduction in the Ca(2+) buffering power of myoplasm in cut fibers and an increase in the flux of Ca(2+) during release.  相似文献   

19.
It is well known that the force developed by skeletal muscles increases with temperature. Despite the work done on this subject, the mechanism of force potentiation is still debated. Most of the published papers suggest that force enhancement is due to the increase of the individual cross-bridge force. However, reports on skinned fibers and single-molecule experiments suggest that cross-bridge force is temperature independent. The effects of temperature on cross-bridge properties in intact frog fibers were investigated in this study by applying fast stretches at various tension levels (P) on the tetanus rise at 5 degrees C and 14 degrees C to induce cross-bridge detachment. Cross-bridge number was measured from the force (critical force, P(c)) needed to detach the cross-bridge ensemble, and the average cross-bridge strain was calculated from the sarcomere elongation needed to reach P(c) (critical length, L(c)). Our results show that P(c) increased linearly with the force developed at both temperatures, but the P(c)/P ratio was considerably smaller at 14 degrees C. This means that the average force per cross bridge is greater at high temperature. This mechanism accounts for all the tetanic force enhancement. The critical length L(c) was independent of the tension developed at both temperatures but was significantly lower at high temperature suggesting that cross bridges at 14 degrees C are more strained. The increased cross-bridge strain accounts for the greater average force developed.  相似文献   

20.
Longitudinal impedance of single frog muscle fibers   总被引:2,自引:2,他引:0       下载免费PDF全文
The longitudinal impedance of single skeletal muscle fibers has been measured from1 to 10,000 Hz in an oil gap apparatus which forces current to flow longitudinally down the fiber. The impedance observed is purely resistive in some fibers from the semitendinosus muscle and in two fibers from the sartorius muscle. In other fibers from the semitendinosus muscle a small phase shift is observed. The mean value of the maximum phase shift observed from all fibers is 1.07 degrees. The artifacts associated with the apparatus and method are examined theoretically and it is shown that one of the likely artifacts could account for the small phase observed. It is concluded that the longitudinal impedance of skeletal muscle fibers is essentially resistive and that little, if any, longitudinal current crosses the membranes of the sarcoplasmic reticulum.  相似文献   

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