The control of energy release in extracted muscle fibers

Tension and P liberation were determined at the same time in glycerol-extracted muscle fibers suspended in ATP solutions. In the relaxed state, produced by ATP in rather fresh preparations, P liberation was low, but somewhat higher than in normal resting muscle. On addition of small amounts of CaCl₂ the fibers gave a strong contraction during which P liberation was on the average about 5 times higher than in the relaxed condition. In aged muscle fibers ATP always produced a strong contraction associated with a high ATPase activity which was not influenced by Ca. The P liberation during a sustained contraction was much smaller in extracted fibers than in normal muscle, but the former maintained tension much more economically than the latter, resembling smooth muscle in this respect. Also the removal of Mg caused a contraction associated with high ATPase activity. Mg, therefore, is inhibitory in relaxed fibers. In fibers activated by Ca or by aging, however, it caused enhancement. The effects of ions on ATPase activity of relaxed fibers are similar to those on myosin and dissociated actomyosin, whereas activated fibers resemble actomyosin at low salt concentration.     

Light and electron microscopic study of adenosine triphosphatase activity of anuran tadpole musculature.

The histochemical activities of succinic dehydrogenase (SDH) and Ca++-activated ATPase (pHs 7.4 and 9.4) were studied in the larval tail musculature of Rana japonica, Rana catesbeiana and Rana ornativentris. The ATPase reaction product was detected by both light and electron microscopy. 'Red' and 'white' muscle fibres, as distinguished by SDH, showed high and low Ca++-ATPase reaction, respectively, at pHs 7.4, 9.4 and following preincubation in cold K2-EDTA solution. The ultrastructural investigation of Ca++-ATPase reaction at pH 7.4 by the Ca++-citrophosphate technique demonstrated electron-dense reaction product in association with A, I and 'Z' bands, intermyofibrillar (SR) compartment and the mitochondrial inner chamber. However, Pb++ precipitation technique demonstrated Mg++-activated myosin ATPase activity at pH 9.2 ultrastructurally. The present histochemical data suggest that the anuran larval tail 'red' muscle fibres are possible 'slow,' and emphasize a possible lack of correlation between the speed of contraction with their ATPase activity. Moreover, 'red' muscle fibres of the anuran tai- musculature are not equivalent to 'Type I' fibres of higher chordates.     

Intrafusal fibre types in rat limb muscle spindles

Summary Morphological, histochemical and ultrastructural characteristics of intrafusal fibre types were studied in rat muscle spindles. The existence of three intrafusal fibre types, namely the typical bag, the intermediate bag and the chain fibres was confirmed. Intrafusal fibres differ in diameter, length and number of nuclei in the equatorial zone. Histochemically, typical bag fibres exhibit both alkali-and acid-stable ATPase activity and low SDH activity. Intermediate bag fibres possess low alkali-stable ATPase activity; after acid-preincubation, however, they have low activity only in the juxtaequatorial region, whereas in the polar zones they exhibit high acid-stable ATPase activity. The SDH activity varies from moderate to high. The chain fibres exhibit high alkali-stable and low acid-stable ATPase and high SDH activity in the extensor digitorum longus muscle, whereas in the soleus muscle the acid-stable ATPase activity varies from a low one to a high one, either among individual chain fibres in one spindle, and/or repeatedly along the fibre length.Since there are regional differences in morphological characteristics and in staining properties of intrafusal fibres, a reliable identification of intrafusal fibre types can only be achieved by an analysis of serial sections.     

Kinetic studies on the initial contraction dependent high ATPase activity of actomyosin molecules

In contracting (superprecipitating) clearing and fully contracted (previously superprecipitated) actomyosin molecules the presteady state phosphate burst was found to be 2 nanomoles inorganic phosphate (Pi) per nanomole myosin. In these muscle models a significant difference in the Mg2+ ATPase activity was found following the initial phosphate burst. Between 120 and 800 milliseconds after the commencement of the reaction the Mg2+ ATPase activity of contracting actomyosin molecules was 5-10 times greater than that of the fully contracted or clearing actomyosin molecules. In the same time interval the rate of turbidity increase of the contracting actomyosin molecules was about 10 fold greater than during the remainder of the time to reach maximal superprecipitation. This high initial ATPase activity found to be present only in the contracting actomyosin molecules and coinciding with the high rate of the velocity of contraction provides sufficient energy for contraction. We propose that this high Mg2+--ATPase activity following the initial burst and included as a part of "conventional" steady state ATPase activity is the source of energy for muscular contraction. Calculation of kinetic and thermodynamic constants indicates that the contracting actomyosin molecule is subjected to a conformational change. As a consequence of contraction the complementarity of the enzyme site to the intermediate complex decreases about 100 fold. Thus the contracted molecules temporarily become relatively refractive to provide energy for the contractile process. In our opinion these findings are important with regard to muscular contraction.     

Intrafusal fibre types in rat limb muscle spindles: morphological and histochemical characteristics.

Morphological, histochemical and ultrastructural characteristics of intrafusal fibre types were studied in rat muscle spindles. The existence of three intrafusal fibre types, namely the typical bag, the intermediate bag and the chain fibres was confirmed. Intrafusal fibres differ in diameter, length and number of nuclei in the equatorial zone. Histochemically, typical bag fibres exhibit both alkali- and acid-stable ATPase activity and low SDH activity. Intermediate bag fibres possess low alkali-stable ATPase activity; after acid-preincubation, however, they have low activity only in the juxtaequatorial region, whereas in the polar zones they exhibit high acid-stable ATPase activity. The SDH activity varies from moderate to high. The chain fibres exhibit high alkali-stable and low acid-stable ATPase and high SDH activity in the extensor digitorum longus muscle, whereas in the soleus muscle the acid-stable ATPase activity varies from a low one to a high one, either among individual chain fibres in one spindle, and/or repeatedly along the fibre length. Since there are regional differences in morphological characteristics and in staining properties of intrafusal fibres, a reliable identification of intrafusal fibre types can only be achieved by an analysis of serial sections.     

Calcium/calmodulin regulation of ATPase activity and endogenous phosphorylation of mammalian brain actomyosin

Rabbit brain actomyosin showed several fold stimulation of the MgATPase activity by Ca2+ alone and by Ca2+/calmodulin. The calmodulin-binding drug, fluphenazine, abolished the stimulated activity. In the presence of Ca2+, exogenous calmodulin had a biphasic effect on ATPase activity at low concentrations (less than 0.15 microM) and activated the ATPase activity by 60-70% at about 1 microM. Tropomyosin-troponin complex from skeletal muscle did not stimulate the ATPase activity of brain actomyosin, but conferred Ca2+ sensitivity to a skeletal muscle myosin/brain actomyosin mixture. These results indicate the presence of myosin-linked, calmodulin-dependent, Ca2+-regulatory system for brain actomyosin. Heavy and light chains of brain myosin were found to be rapidly phosphorylated by endogenous Ca2+-dependent protein kinase(s). Ca2+-independent phosphorylation of one of the light chains was also observed.     

ATPase Activity of Myosin Correlated with Speed of Muscle Shortening

Myosin was isolated from 14 different muscles (mammals, lower vertebrates, and invertebrates) of known maximal speed of shortening. These myosin preparations were homogeneous in the analytical ultracentrifuge or, in a few cases, showed, in addition to the main myosin peak, part of the myosin in aggregated form. Actin- and Ca⁺⁺-activated ATPase activities of the myosins were generally proportional to the speed of shortening of their respective muscles; i.e. the greater the intrinsic speed, the higher the ATPase activity. This relation was found when the speed of shortening ranged from 0.1 to 24 muscle lengths/sec. The temperature coefficient of the Ca⁺⁺-activated myosin ATPase was the same as that of the speed of shortening, Q₁₀ about 2. Higher Q₁₀ values were found for the actin-activated myosin ATPase, especially below 10°C. By using myofibrils instead of reconstituted actomyosin, Q₁₀ values close to 2 could be obtained for the Mg⁺⁺-activated myofibrillar ATPase at ionic strength of 0.014. In another series of experiments, myosin was isolated from 11 different muscles of known isometric twitch contraction time. The ATPase activity of these myosins was inversely proportional to the contraction time of the muscles. These results suggest a role for the ATPase activity of myosin in determining the speed of muscle contraction. In contrast to the ATPase activity of myosin, which varied according to the speed of contraction, the F-actin-binding ability of myosin from various muscles was rather constant.     

State of the contractile apparatus in the development of a pathological process in muscle. IV. Effect of Ca2+ on the process of contraction nodule formation and on the ATPase activity of muscle actomyosin in Zenker's necrosis

Using phase-contrast and polarized ultraviolet (UV) fluorescent microscopy, the structure of single muscle fibres was studied in the course of the contraction module formation during Zenker's necrosis. The degree of manifestation of destructive changes in the contractile system was shown to depend upon the concentration of extracellular Ca-ions. With decreasing Ca2+ concentration, the fibre loses the ability to form contraction nodules peculiar to the Zenker necrosis, and the development of this process is interrupted at the stage of sarcomere supercontraction. The UV fluorescent anisotropy pattern of fibre regions, conforming with the contraction nodules, suggests the occurrence of a more pronounced disorganization of contractile system in the presence of Ca2+. The ATPase activity of actomyosin isolated from altered muscle was studied to appreciate the functional state of the contractile system. This actomyosin was found to be inactivated 1.5 times as much as that isolated from muscles treated during Zenker's necrosis in calcium-free media.     

Effect of nitric oxide on ATPase activity of smooth muscle actomyosin

The effects of nitric oxide donor sodium nitroprusside (SNP) on ATPase activities of smooth muscle actomyosin and myosin were investigated. The effect of SNP on actomyosin ATPase activity was biphasic: the low concentration of this reagent increased the actomyosin ATPase activity while the high concentration exerted opposite effect. These effects were similar to those induced by the specific thiol-alkylating agent N-ethylmaleimide. These data demonstrate that nitric oxide exert the direct effect on smooth muscle contractile proteins. Such effect may be involved in physiological action of NO on smooth muscle.     

Cross-bridge model of muscle contraction. Quantitative analysis

We recently presented, in a qualitative manner, a cross-bridge model of muscle contraction which was based on a biochemical kinetic cycle for the actomyosin ATPase activity. This cross-bridge model consisted of two cross-bridge states detached from actin and two cross-bridge states attached to actin. In the present paper, we attempt to fit this model quantitatively to both biochemical and physiological data. We find that the resulting complete cross-bridge model is able to account reasonably well for both the isometric transient data observed when a muscle is subjected to a sudden change in length and for the relationship between the velocity of muscle contraction in vivo and the actomyosin ATPase activity in vitro. This model also illustrates the interrelationship between biochemical and physiological data necessary for the development of a complete cross-bridge model of muscle contraction.     

Histochemical heterogeneity of intrafusal muscle fibres in slow and fast skeletal muscles of the rat

Summary Intrafusal muscle fibres of the slow soleus (Sol) and fast vastus lateralis (VL) muscles of the rat were studied histochemically. Serial transverse sections were incubated for the localization of succinate dehydrogenase (SDH), alpha glycerophosphate dehydrogenase (GPD) and adenosine triphosphatase (ATPase). The latter was examined further after preincubation in acidic solution held at either low or room temperature (R_T). The bag₂ intrafusal fibres in both muscles displayed high regular and acid stable ATPase, but low SDH and GPD activities. Bag₁ intrafusal fibres showed low to moderate regular ATPase, a regional heterogeneity after R_T acid preincubation (low activity in juxtaequatorial and high in polar zones), moderate SDH, but low GPD reactions. In both muscles the chain fibres usually exhibited high ATPase for both regular and cold acid preincubated reactions, but usually low activity after R_T acid preincubation; they had high SDH but variable GPD activities. In Sol muscle, however, approximately 25% of spindles contained chain fibres that showed high acid-stable ATPase reaction after both cold and R_T acid preincubation. In contrast, chain fibres in some VL spindles had a characteristically low ATPase reaction even after cold acid preincubation. This study, therefore, has delineated the existence of an inherent heterogeneity among chain fibres (with respect to their histochemical reactions) in muscle spindles located within slow and fast muscles and also between those found within populations of either Sol or VL muscle spindles.     

Sensitivity of actomyosin ATPase to calcium and strontium ions. Effect of hybrid troponins

1. Hybrid or reconstituted troponins were prepared from troponin components of rabbit skeletal muscle and porcine cardiac muscle and their effect on the actomyosin ATPase activity was measured at various concentrations of Ca2+ or Sr2+. The Ca2+ concentration required for half-maximum activation of actomyosin ATPase with troponin containing cardiac troponin I was slightly higher than that with troponin containing skeletal troponin I. The Sr2+ concentration required for half-maximum activation of actomyosin ATPase with troponin containing skeletal troponin C was higher than that with troponin containing cardiac troponin C. 2. Reconstituted cardiac troponin was phosphorylated by cyclic AMP-dependent protein kinase. The Ca2+ sensitivity of actomyosin ATPase with cardiac troponin decreased upon phosphorylation of troponin I; maximum ATPase activity was depressed and the Ca2+ concentration at half-maximum activation increased. On the other hand, phosphorylation of troponin I did not change Sr2+ sensitivity. 3. The inhibitory effect of cardiac troponin I on the actomyosin ATPase activity was neutralized by increasing the amount of brain calmodulin at high Ca2+ and Sr2+ concentrations but not at low concentrations. 4. ATPase activity of actomyosin with a mixture of troponin I and calmodulin was assayed at various concentrations of Ca2+ or Sr2+. The Ca2+ or Sr2+ sensitivity of actomyosin ATPase containing skeletal troponin I was approximately the same as that of actomyosin ATPase containing cardiac troponin I. Phosphorylation of cardiac troponin I did not change the Ca2+ sensitivity of the ATPase. 5. The Ca2+ or Sr2+ concentration required for half-maximum activation of actomyosin ATPase with troponin I-T-calmodulin was higher than that of actomyosin ATPase with the mixture of troponin I and calmodulin. Maximum ATPase activity was lower than that with the mixture of troponin I and calmodulin.     

Kontraktionseigenschaften von isoliertem Schleimpilzactomyosin

Summary The isolated contractile proteins of the slime mouldPhysarum polycephalum and of rabbit skeletal muscle were investigated by using actomyosin thread models. The actomyosins were compared with respect to contraction behaviour, fine structure, and ATPase activity. Thread models were made of natural and synthetic actomyosins of both systems.The natural actomyosins differ considerably: The actin filament length ofPhysarum actomyosin is only about one fourth, the ATPase activity and actin/myosin ratio are much lower compared to natural muscle actomyosin. The contraction rate of the natural slime mould actomyosin is remarkably slower than that of the natural muscle actomyosin.The synthetic actomyosins were formed from separately isolated actins and myosins with a constant actin/myosin ratio and comparable actin filament lengths. The thread models of either recombined and hybridized actomyosins of both systems contract with nearly identical rates. The comparison of the synthetic actomyosins shows that under comparable conditions a) the actomyosins of both systems perform work with the same efficiency, b) the actin and myosin component is freely exchangeable without any change in the rate of actomyosin contraction. These results indicate that in both skeletal muscle and slime mould the force generation is based on the same mechanism of actin-myosin interaction.

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Ein Teil dieser Ergebnisse wurde als Symposiumsvortrag auf dem 9th Meeting of the Federation of the European Biochemical Societies, Budapest vorgetragen.     

The ultrahistochemical picture of the so-called reversed ATPase in the gastrocnemius muscle of the rat.

The ultrahistochemical localization of the "reversed" ATPase activity was investigated. Red muscle fibres showed permanent sarcomere contraction, enzymatic activity in the inner membrane and matrix of mitochondria, and large, osmiophilic, probably calcium-containing structures within mitochondria and on their outside. White muscle fibre sarcomeres were relaxed, and activity within their sarcoplasmic reticulum was marked, but slight in the mitochondria. The relaxed state of the sarcomere in the white muscle fibres is supposed to be connected with inactivation of myofibrillar ATPase by acid preincubation, whereas red muscle contraction indicates that acid preincubation does not inactivate their myofibrillar ATPase. That the product of its activity failed to become visible in the sarcomeres is probably due to imperfection of the method. Two sub-types of red muscle fibres were distinguished: those showing only enzymatic activity in mitochondria, and those containing large intra- and extramitochondrial osmiophilic structures. The origin and composition of these structures is difficult to explain. A relation seems to exist between their presence within mitochondria and outside.     

Actin-binding protein amplifies actomyosin contraction,and gelsolin confers calcium control on the direction of contraction

Cross-linking of muscle actin filaments by low concentrations of actin-binding protein reduces the concentration of muscle myosin required for contraction of actin. Gelsolin, a macrophage protein that divides actin filaments in the presence of calcium, inhibits the amplifying effect of actin-binding protein on contraction of actomyosin. In a calcium gradient, the actomyosin gel moves from high to low calcium concentrations, indicating that calcium-controlled lattice formation can impart directionality to the movement of an isotropic actin network.     

The effect of hydrostatic pressure on the interaction of actomyosin subfragment 1 with nucleotides.

Increased hydrostatic pressure has previously been shown to reduce the tension of isometrically contracting skinned muscle fibres. An isomerization of the actomyosin complex is known to be pressure sensitive, but the pressure sensitivity of other steps in the ATPase pathway has not been characterised. We report here the effect of pressure on the ATP hydrolysis step of the myosin subfragment 1 ATPase, ADP binding to actomyosin subfragment 1 and the rate of ATP induced dissociation of actomyosin subfragment 1. We discuss the relationship of these changes to the observed effect of pressure on skinned muscle fibres.     

Oxygen exchange reaction during ATP hydrolysis by glycerinated muscle fibers, myofibrils, and synthetic actomyosin filaments

The oxygen exchange during ATP hydrolysis by glycerinated muscle fibers, myofibrils, and synthetic actomyosin filaments was studied from the distribution of the [18O]Pi species produced by the hydrolysis of [gamma-18O]ATP. The products were mixtures of two species, one with a low extent of oxygen exchange and the other with a high extent. The low and high extents of oxygen exchange in these two Pi species were the same as those of the acto-S-1 ATPase reaction through the routes with and without the dissociation of actomyosin, respectively (Yasui, M., Ohe, M., Kajita, A., Arata, T., & Inoue, A. [1988] J. Biochem. 104, 550-559). During isometric contraction of glycerinated muscle fibers at 20 degrees C, the fraction of ATP hydrolysis with low extent of oxygen exchange was 0.83 and 0.70, respectively, in 0 and 120 mM KCl. In myofibrils, the fraction of ATP hydrolysis with a low extent of oxygen exchange was 0.72-0.88 in 0-120 mM KCl at 20 degrees C. Therefore, in glycerinated muscle fibers and myofibrils ATP seems to be mainly hydrolyzed through a route without the dissociation of actomyosin, especially at low ionic strength and at room temperature when the tension development is high. ATP hydrolysis through this route may be coupled with muscle contraction.     

Effect of ethanol on tropomyosin in solution and in reconstituted thin filaments

The effects of ethanol at concentrations below 10% on the conformation of tropomyosin, its end-to-end polymerization, its binding to F-actin, and its effects on actomyosin ATPase activity were studied. Ethanol stabilized the tropomyosin conformation by shifting the helix thermal unfolding profile to higher temperatures, and increased the end-to-end polymerization of tropomyosin. Ethanol-induced changes in the excimer fluorescence of pyrene-tropomyosin indicated that its conformation was stabilized by ethanol both free and bound to F-actin. Effects of tropomyosin and tropomyosin-troponin on actomyosin ATPase activity were measured under conditions for which tropomyosin binding to F-actin increases the activity. Under conditions for which the binding of tropomyosin to F-actin is optimum, in the presence of tropomyosin, the actomyosin ATPase activity decreased as the ethanol concentration increased, further indicating that ethanol induces a structural change in the tropomyosin-F-actin complex. Under conditions for which the binding of tropomyosin to F-actin is weak (low salt or high temperature), addition of ethanol increased the ATPase activity due to increased binding of tropomyosin to F-actin. Thus, ethanol appears to modify actomyosin ATPase activity by increasing the binding of tropomyosin to F-actin and affecting the structure of tropomyosin in the tropomyosin-F-actin filament.     

Association and dissociation of the thick and thin filaments within myofibrils in conditions of “contraction” and “relaxation”

1. The current assumption that the low ATPase activity of relaxed myofibrils is represented by the ATPase activity of myosin which has been set free during the dissociation of actomyosin was investigated. For this purpose, the ATPase activity of relaxed skeletal myofibrils of the rabbit and of the crab Maia squinado has been compared with the activity of contracted fibrils and of purified rabbit myosin in conditions of varying ionic strength, pH and concentrations of MgATP (i.e. MgATP²⁻ + MgHATP⁻) and Mg²⁺.

2. Contraction and relaxation of the fibrils was induced by changing the concentration of Ca²⁺ from about 5×10⁻⁵ to below 1×10⁻⁸ M.

3. In all conditions studied, the ATPase activity of relaxed fibrils was about 6–8 times less than that of the contracted fibrils, but it remained a typical actomyosin ATPase.

4. Quantitatively and qualitatively, this ATPase differs from the ATPase of myosin. For instance, its dependence on pH is the reverse of that of the myosin ATPase.

5. Calculation showed that the fibrils are dissociated by 90% in conditions of relaxation. Since the ATPase activity of myosin was merely some 2% of the actomyosin activity, the major part of the ATPase of fibrils, even at a dissociation of 90%, is bound to show the properties of the ATPase of actomyosin.

6. However, a dissociation of 90% cannot be distinguished from a dissociation of 100% by means of physical methods (viscosity, superprecipitation, resistance to stretch, etc.). This explains why physical methods indicate a “full” dissociation of actomyosin although, enzymatically, the ATPase is still of the actomyosin type.

7. The possible reasons are discussed for the discrepancy between the 100-fold increase in the ATP turnover and the 1000-fold increase in energy turnover of the living muscle during the transition from relaxed to active state. The most probable explanation seems to be an ATPase activity of myosin which is too high by a factor of ten as compared to the energy turnover of living muscle at the resting state. This high activity cannot be caused by a contamination of the myosin by Ca²⁺-insensitive actomyosin.     

pH lability of myosin ATPase activity permits discrimination of different muscle fibre types in crustaceans

Myofibrillar actomyosin ATPase activity has been studied histochemically in the closer muscle of the crab Eriphia spinifrons. Preincubation at pH 4.6 and 5.0 reveals differences in the lability of the ATPase. This permits the discrimination of four fibre types. Of these, three represent subgroups of rapidly contracting fibres. The histochemically defined fibre types correspond well with four groups defined according to electrophysiological criteria.