Nature of the changes in the activity of water-soluble enzymes in exposure of muscles to harmful agents. IV. The study of hexokinase extractability from intact and altered muscles

A possibility of hexokinase binding with actomyosin in skeletal muscles of Rana temporaria L., and the effect of thermal alteration (15 min at 36, 37, 38, 40 and 42 degrees C) on the binding were studied. Solutions of KCl (0.075 M and 0.15 M) extract more hexokinase from intact and altered muscles than does an non-electrolyte medium. Hexokinase freely dissolved in hyaloplasm is extracted in non-electrolyte medium. Hexokinase bound with structural components of the muscle cell is extracted upon the increase in ionic force of the extractant. The solubilizing effect of electrolytes on hexokinase is higher in alterated muscles than in the intact muscles indicating the increase in hexokinase binding under thermal alteration. Actomysin isolated from muscles reveals hexokinase activity. In reprecipitated actomyosin, the larger part of its hexokinase remains in actomyosin gel, the level of hexokinase activity not depending on the number of reprecipitation procedures or on the volume of washing solution. Hexokinase in actomyosin gel is less stable to the thermal action than in water supernatant of muscle extract. This may be due to the increase in hexokinase binding with actomiosin whose sorption activity increases under the thermal denaturation.     

The nature of the changes in the activity of water-soluble enzymes in action on the muscles of damaging agents. VI. Glyceraldehyde-3-phosphate dehydrogenase binding and enzyme adsorption by F-actin in ghost muscle fiber]

A possibility of binding glyceraldehyde-3-phosphate dehydrogenase (GAPhDG) in frog (Rana temporaria L.) skeletal muscles was studied by measuring its solubilization in 0.15 M KCl and by its presence in isolated actomyosin. Using a 0.15 M KCl solution, more GAPhDG was extracted from intact muscles and muscles treated with heat at 38, 42 and 46 degrees C for 15 min than in a non-electrolyte medium. Actomyosin isolated from muscles reveals GAPhDG activity which cannot be removed by actomyosin reprecipitation. In myosin-, troponin- and tropomyosin-free single glycerinated muscle fibres (ghost fibres) GAPhDG absorption to F-actin was shown. It is suggested that under thermal injure of muscle cells, the increase in GAPhDG binding with thermolabile proteins of actomyosin complex may occur.     

The nature of changes in the activity of water-soluble enzymes during the action of injurious agents on the muscles. V. A study of the extractability of lactate dehydrogenase from intact and altered muscles

A study was made of lactate dehydrogenase (LDH) extractability from intact and thermally injured muscles of Rana temporaria L. in the 0.15 M KCl solution and in the non-electrolyte medium. A 15 minute incubation of intact muscles and those treated with heat at 38, 42, 44 and 46 degrees C in the 0.15 M KCl solution led to a much higher extraction of LDH than in the non-electrolyte medium. Following heating at 38 degrees C, causing irreversible injury of muscles, the LDH extractability in the non-electrolyte medium is seen to fall, whereas in the 0.15 M KCl solution it remained at the same level as after LDH extraction from intact muscles. The decrease in LDH extractability in non-electrolyte medium from thermally injured muscles may be due to the increase in LDH binding with thermolabile structural components of muscle. One of the components of muscle cell, known to bind LDH, is actomyosin, since it is isolated from muscles together with LDH, which cannot be removed by actomyosin reprecipitation or by increasing the volume of washing solution.     

Changes in phosphofructokinase activity upon exposure of muscles and muscle extracts to injurious agents

A study has been made on changes of outflux, extractability and activity of phosphofructokinase (PPK) under the action of heating, and of urea on the frog's skeletal muscles and on their extracts. Under the action of heating on muscles, the decrease of PPK activity (to 35%) is first revealed 34--36 degress C, when decrease of excitability and the contracture are not yet detected, and the extractability of the total water-soluble protein does not change. At the start of contracture, and at the decrease of excitability (at 38 degrees C) PPK in the muscle loses its activity. The thermolability of PPK is the greatest one compared to all the enzymes investigated before. The data on the high thermolability of PPK are discussed in terms of the regulating role of PPK in glycolysis. The PPK activity of extracts of muscles altered by urea, during the action not accompanied by the death of muscles, does not change. At the irreversible disappearance of muscle excitability, PPK is inactivated. PPK in the cell is more stable to injuring agents than PPK in isolated state.     

Dependence of energy transduction in intact skeletal muscles on the time in tension.

In intact single crayfish muscle fibers and frog semitendinosus muscles we have studied the tension response to sinusoidal length changes in the frequency range of 0.25-133 Hz. By this method we have resolved three processes in the interaction of myosin cross-bridges with actin in fully activated preparations. They are (A) a low-frequency phase advance, (B) a middle-frequency delay, and (C) a high-frequency advance. These processes can be used as probes to study the chemomechanical coupling of contractility. Process (B) represents net power output from the muscle preparation (oscillatory work). With maximal K or caffeine activation of crayfish muscle at 20 degrees C, it decreases to zero in the initial 45 s of maintained tension. Similar results were obtained with frog semitendinosus whole muscles. We interpret this decrease of (B) with time as a gradual decrease in actomyosin ATP-hydrolysis rate.     

The comparative characteristics of myofibrillar protein transformation in muscles of different phenotypes

Studies have been made on Mg2(+)-ATPase, Ca2(+)-sensitivity, superprecipitation and fractional composition of natural and desensitized actomyosin from myocardium, slow and fast skeletal muscles after physical training (swimming, gravitational loading) and after monthly readaptation. Physical overloading makes physicochemical properties and protein composition of actomyosin from the myocardium and slow skeletal muscles similar to those in fast skeletal ones. Changes in actomyosin from the myocardium and slow skeletal muscles are more profound, whereas the recovery of the initial properties during readaptation reveals high plasticity of muscles of these phenotypes. Changes in Ca regulation depend mainly on muscle phenotype. Different plasticity of muscles of various phenotypes during readaptation results from differences in the synthesis of protein components of myofibrils.     

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.     

Calcium ion-dependent myosin from decapod-crustacean muscles.

Ca2+ regulation of arthropod actomyosin adenosine triphosphatase is associated with both the thin filaments, as in vertebrates, and with the myosin, as in molluscs. The actomyosin of decapod-crustacean fast muscles was previously considered to be an exception, displaying only a Ca2+-regulatory system linked to the thin filaments and not a myosin-linked regulatory system. In the present study, myosin regulation is demonstrated in a variety of decapod muscles when they are tested under more physiological ionic conditions. Myosin regulation is shown by using mixtures of pure rabbit actin with myofibrils, with actomyosin and with purified myosin, and in each case the adenosine triphosphatase is Ca2+ dependent. Myosin regulation may also occur in vertebrate striated muscle, but seemingly is lost during purification of the myosin.     

An activating factor (tropomyosin) for the superprecipitation of actomyosin prepared from scallop adductor muscles

An activating factor for the superprecipitation of actomyosin reconstructed from scallop smooth muscle myosin and rabbit skeletal muscle F-actin was purified from thin filaments of scallop smooth and striated muscles. Two components were obtained from the smooth muscle and one from the striated muscle. All three components similarly affected the actomyosin ATPase activity. According to the results of analysis involving double reciprocal plotting of the ATPase activity versus F-actin concentration, the activating factor for superprecipitation decreased the apparent dissociation constants of actomyosin about 30 to 110 times. The activation of the superprecipitation by the factor, therefore, may be due to the enhancement of the affinity between F-actin and myosin in the presence of ATP. The activating factor was identified as tropomyosin based on it mobility on polyacrylamide gel electrophoresis and on the recovery of the Ca2+-sensitivity of purified rabbit skeletal actomyosin in the presence of troponin.     

A histoenzymatic study of rat intrafusal muscle fibres.

The histochemical activities of myofibrillar adenosine triphosphatase (ATPase), succinic dehydrogenase (SDH) and alpha glycerophosphate dehydrogenase (alpha-GPD) were studied in intrafusal muscle fibres of rat fast and slow muscles. The ATPase reaction was carried out after the three standard acid preincubations. The cold K2-EDTA preincubated ATPase reaction product was similar to that seen following the regular or alkali-preincubated ATPase reaction, except that the intermediate bag fibres exhibited much higher activity after cold K2-EDTA preincubation. Following either acetic acid solution or cold and room temperature K2-EDTA-preincubation, followed by the ATPase reaction, chain fibres of the fast muscles vastus lateralis and extensor digitorum longus exhibited a very low amount of reaction product as compared with those of the slow soleus. Veronal acetate and K2-EDTA preincubations (and equally preincubation in acetic acid solution) resulted in acid stable ATPase activity along the entire length of the typical bag fibres but only in the polar regions of the intermediate bag fibres. On the basis of differing alpha-GPD reaction, two sub populations of nuclear chain fibres were discovered in one spindle. It is a matter of conjecture, to what extent the histochemical differences of intrafusal fibres from fast and slow muscles reflects functional distinctions in the response to stretch of muscle spindles from fast and slow muscles.     

Histochemical mapping and fiber size analysis of mimic muscles.

Fourteen functionally relevant mimic muscles of nine human bodies were analyzed with respect to their muscle fiber sizes and their histochemical fiber type composition. In cryostat sections stained for actomyosin ATPase, type 1 and type 2 fibers were evaluated separately by means of computer-assisted image analysis. The fiber diameters varied between 20.24 and 41.45 microns. According to the proportions of the fiber types, the mimic muscles could be classified into three groups: (1) phasic muscles, with 14 to 15 percent type 1 fibers, (2) intermediate muscles, with 28 to 37 percent type 1 fibers, and (3) tonic muscles, containing 41 to 67 percent type 1 fibers. It is concluded that one has to consider this diversity of mimic muscles when planning the surgical reconstruction of facial paralysis.     

Comparison of the effect of castration on the development of postural and non-postural muscles of mice.

The effect of castration on the development of muscle mass of postural and non-postural muscles was studied in 18 male mice (9 castrated, 9 uncastrated). Results obtained indicated that the castrated males grew faster and were bigger in body size and weight at maturity than the intact males. The bigger body size of castrated males was not due to larger muscle mass but was probably due to increased subcutaneous fat deposition. Atrophy of muscles usually observed following castration was significantly greater in the non-postural (biceps brachii) muscle of the forelimb as compared to the postural (triceps brachii) muscle of the forelimb. Conversely, the amount of reduction in muscle mass was similar in both postural (soleus) and non-postural (tibialis cranialis) muscles of the hindlimb.     

Comparison of the myosin and actomyosin ATPase mechanisms of the four types of vertebrate muscles

The mechanism of ATP hydrolysis by myosin and actomyosin was investigated for the four major classes of vertebrate muscles: fast white (posterior latissimus dorsi), slow red (anterior latissimus dorsi), cardiac and smooth (gizzard). The kinetic behavior of all classes of muscle was consistent with the scheme developed previously for rabbit fast white muscle, but quantitative differences were observed for the rate constants of some of the steps in the hydrolysis cycle. The rate of the hydrolysis step of myosin subfragment-1 was similar for the striated muscles and two to three times smaller for smooth muscle. Two isomerizations of the enzyme occurred in the pathway leading to the formation of the myosin-products intermediate. The rate of dissociation of acto S–1 by ATP was slower for slow muscles and a maximum rate was observed at low temperature. The rate of association of the S-1-products intermediate with actin was equal to the turnover rate of acto S–1 ATPase at low concentrations of actin. The rate of dissociation of ADP from an acto S–1-ADP complex was also much slower for slow muscle. It was shown by Barany (1967) that the maximum turnover rate of actomyosin ATPase (V_M) is proportional to the velocity of contraction of the muscle. The only step in the mechanism that is correlated with V_M is the apparent second-order rate constant for the formation of a complex of the S-1-product state with actin. The evidence is discussed in terms of a mechanism in which the release of reaction products from actomyosin is the step that is of primary importance in determining the value of V_M and the velocity of contraction.     

Comparison of fiber types in skeletal muscles from ten animal species based on sensitivity of the myofibrillar actomyosin ATPase to acid or copper

Comparisons were made of the histochemical characteristics of skeletal muscle from 10 animal species. The basic comparison was made from the staining patterns for the myofibrillar actomyosin ATPase produced by preincubation of fresh frozen cross-sections of muscle at alkaline pH (10.30) or acid pH (4.60) with those produced by preincubation in media containing Cu2+ at alkaline pH (10.30), near neutral pH (7.40), or acid pH (4.60). Muscle sections were also stained for reduced nicotinamide adenine dinucleotide tetrazolium reductase and alpha-glycerophosphate dehydrogenase to provide an indication of the relative oxidative and glycolytic capacity of the different fiber types. Type II fibers in mixed fibered muscles were either very sensitive, moderately sensitive, or relatively insensitive to inactivation of the myofibrillar actomyosin ATPase after acid preincubation. These fibers were identified as type IIA1, IIA2, and IIA3, respectively. The myofibrillar actomyosin ATPase of the type I fibers of these muscles, with the exception of those in mouse muscle, was activated by pretreatment with acid. A separation of animal species was possible based on the stability of the IIA1 fibers to inclusion of Cu2+ in the preincubation medium. For one group of animals (rat, mouse, monkey, man, dog, rabbit, and cow), a reciprocal relationship existed between lability to acid and stability to Cu2+ for type IIA1 and IIA3 fibers, respectively. For the second group of animals (horse, ass, and cat) there was a parallel relationship between lability or stability of the type IIA1 and IIA3 fibers to pretreatment with either acid or Cu2+.     

Some properties of transport ATPases in functionally different muscles]

The properties of Ca-transporting system in sarcoplasmic reticulum membranes in fast and slow frog muscles as well as some properties of sarcolemma Na, K-ATPase of the same object were investigated. The rate of Ca2+ uptake, Ca-ATPase activity and Ca/ATP ratio for the reticulum of fast muscle demonstrated higher values than those for the reticulum of slow muscle. The rate of Ca2+ accumulation by the fragments of the rectus reticulum and Ca/ATP ratio were found to decrease under the influence of acetylcholine (0.05-5 mM). The transport system of the sartorius reticulum was found to be less sensitive to acetylcholine. The peak activity of Na, K-ATPase in femoral muscles of the frog occurred at 80 mM NaCl and 60 mM KCl, whereas in the rectus abdominal muscle it equalled 100 mM NaCl and 40 mM KCl. Thus, Na, K-ATPase activity in the slow muscle was predominantly higher than that in the mixed (femoral) muscles. If the sarcolemma preparations of the muscles of both types the inhibitory effect of acetylcholine on Na; K-ATPase was registered. The enzyme of slow muscles exhibited higher sensibility to acetylcholine.     

Characteristics of functioning of electromechanical coupling in striated muscles of higher and lower vertebrates

A comparative pharmacological analysis of relative contributions of different signal transduction pathways in the activation of contraction (excitation-contraction coupling, ECC) in intact fast striated muscles of frog and lamprey was performed. It was found that the major mechanism responsible for the ECC in muscles of both animals is Ca2+ release from the sarcoplasmic reticulum through the ryanodine-sensitive channels. However, the ECC in lamprey muscle displays some important differences in the units of electromechanical coupling, which precede the calcium release from sarcoplasmic reticulum. The maximum contraction force in frog muscle develops during caffeine-induced contracture, which indicates that all Ca2+ stored in sarcoplasmic reticulum is released through ryanodine-sensitive channels. In contrast, in lamprey muscle, the maximum force develops not in response to high caffeine concentration, but in response to repetitive electrical stimulation. Hence, in addition to stores liberated by ryanodine-sensitive channels, some other sources of calcium ions should exist, which contribute to the contraction activation. A source of this additional Ca2+ ions can be external medium, because acetylcholine contracture is abolished in a calcium-free medium. In frog muscle, the acetylcholine contracture was abolished in a Na(+)-free solution. It was concluded that in frog muscle ECC can be triggered by changes in the transmembrane potential (depolarization-induced calcium release), while in lamprey muscle the entry of calcium ions into myoplasm as the trigger in ECC (calcium-induced calcium release). The lamprey muscle was found to be more resistant to tetrodotoxin and tetracaine, which is indicative of a role in the activation of contraction of tetrodotoxin-resistant Na+ and/or Ca2+ channels. It was concluded, that ECC mechanism in striated muscles of low vertebrates is not limited by the generally accepted scheme of depolarization-induced calcium release but can include some other schemes, which require the Ca2+ influx into the cell.     

The influence of caldesmon on strong binding of myosin with actin in denervated rat skeletal muscles

The effect of caldesmon (CaD) on conformational changes in F-actin modified by fluorescent probe TRITC-phalloidin was investigated by polarized fluorimetry. Changes were induced by a subfragment-1 (S-1) of myosin in the absence or presence of CaD in ghost muscle fibers obtained from intact and denervated slow (SOL) and fast (EDL) skeletal muscles of rats. S-1 binding to actin of both SOL and EDL muscles was shown to cause changes in polarized parameters of TRITC-phalloidin typical for a strong actin-myosin binding as well as of transition ofactin subunits from "off" to "on" state. CaD inhibits this significantly. Denervation atrophy inhibits the effect of S-1 as well but does not affect the capability of CaD decreasing the formation of strong binding in actomyosin complex. It is supposed that CaD "freezes" F-actin structure in "off" state. The denervation atrophy has no effect on CaD responsibility to bind thin filaments and to switch "off" actin monomers.     

Chloride cotransport in the membrane of earthworm body wall muscles

The resting membrane potential (V(m)) of isolated somatic longitudinal muscles of the earthworm Lumbricus terrestris was studied by glass microelectrodes. The inhibition of chloride permeability by low pH did not affect V(m) of the muscle fibers in isolated somatic longitudinal muscles of the earthworm Lumbricus terrestris which was -48.7 mV (inside negative) at pH 7.3 and -49.1 at pH 5.6. On the other hand, bathing the muscles in Cl(-) and Na(+)-free solutions, or application of the chloride transporter inhibitor furosemide and Na(+)-K(+)-ATPase inhibitor ouabain depolarized the V(m) by 3-5 mV. The effects of a Cl(-) -free solution and ouabain were not additive. This demonstrates relatively small contribution of equilibrium potential for Cl(-) to the resting membrane potential and electrogenic effect of Na(+)K(+)-ATPase which is dependent on the supply of Na(+)(i) ions by furosemide-sensitive and Cl(-)(e)- and Na(+)(e)-dependent electroneutral transport (most probably Na(+)K(+)Cl(-) cotransport).     

Tension development of normal and hypertrophied-failing papillary muscles following rapid stimulation

Recent studies have shown that numerous cellular alterations exist in hypertrophied-failing (HF) cardiac muscle. Of particular interest is the finding of an altered ability of the Na-K pump to regulate membrane potential in this tissue during periods of transient stimulation. The present study was designed to determine if this altered Na-K pump function is in any way related to the ability of this tissue to develop force. Along these lines the rate of stimulation (6/min) of normal and hypertrophied-failing right ventricular papillary muscles from cats was increased to 60/min for 90 sec. This procedure was repeated in solutions with low Na+, low Na+ and Ca++, and Ouabain. These solutions were utilized to vary the ionic load on the Na-K pump and the Na-Ca exchanger. The results demonstrate that the pattern of changes in tension in HF papillary muscles seen following periods of rapid stimulation are significantly different from those of normal muscles. The pattern of changes in mechanical performance were found to be similar to the membrane potential changes described in previous studies. In addition, lowering the Na+ load presented to HF muscles returned the characteristic pattern of changes in tension, following drive, toward normal. Ouabain was found to inhibit the changes in tension development following increased rates of stimulation that are thought to be produced by activation of the Na-K pump. The results suggest that the ability of the Na-K pump to maintain normal transmembrane ionic gradients may be altered in HF muscles. This alteration appears to be capable of affecting cellular Ca++ possibly through the Na-Ca exchange system.     

Lipoprotein lipase activity in skeletal muscles of the rat: effects of denervation and tenotomy.

The effects of denervation, tenotomy, or tenotomy with simultaneous denervation on the activity of heparin-releasable and intracellular, residual lipoprotein lipase (LPL) and triacylglycerol (TG) content were examined in rat skeletal muscles. An influence of muscle electrostimulation on denervated and tenotomized muscles was also evaluated. Activity of both LPL fractions was decreased in denervated and/or tenotomized soleus and red portion of gastrocnemius muscles. It was accompanied by a slight elevation of the intracellular TG content. Electrostimulation increased activities of both fractions of LPL in red muscles from intact hindlimbs. In stimulated denervated muscles without or with simultaneous tenotomy, activity of two LPL fractions was also enhanced, but control values were reached only in denervated soleus muscle. Electrical stimulation had no pronounced effect on LPL activity in tenotomized muscles. In conclusion, denervation and/or tenotomy decreases LPL activity in red muscles, indicating reduction of the muscle potential to utilize circulating TG. Electrostimulation only partly restores the diminished LPL activity in denervated muscles, without any effect in tenotomized ones. Thus, to maintain LPL activity in resting muscle, intact innervation and tension are needed.