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.     

Nature of the changes in the activity of water-soluble enzymes as affected by noxious agents acting on the muscles. I. A study of the extractability of phosphofructokinase from intact and altered muscles

Binding of phosphofructokinase (PPK) in intact and thermally altered (15 minutes at 38 degrees C) skeletal muscles of frogs (Rana temporaria) the the extractability of PPK from muscles was studied. PPK activity in actomyosin was also studied. Inhibiting effect of electrolytes (KCl, NaCl, CaCl2, MgCl2) on PPK in muscle extract does not allow to use them for the decision of the question of the interprotein interactions of PPK. 5 mM Na2-EDTA extracts additional PPK from homogenates of intact and altered muscles in comparison with PPK extracted in the media without Na2-EDTA (for intact muscles and altered muscles--509 and 729%). Under alteration of muscle, the binding of PPK increases. Among the structural components of muscle which bind PPK, proteins of actomyosin complex have been found.     

Modification of gizzard myosin and reconstituted actomyosin with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole at low and high ionic strength.

Treatment of reconstituted gizzard actomyosin at 0.15 M or 0.6 M KCl with the fluorescent adenine analog 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, NBD-Cl, resulted in a significant decrease in the labeling of the myosin from actomyosin compared to that of myosin alone. Actin protected partially the K(+)-ATPase activity of myosin from modified actomyosin. The reagent was able to detect changes in the conformation of myosin as the distribution of the label in the heavy and light chains of myosin and actin was different at 0.15 M and 0.6 M KCl. The 6S and 10S transition, unique to smooth muscle myosin, can be monitored with the aid of this reagent.     

AMP and IMP dissociate actomyosin into actin and myosin

We investigated to determine why heating of squid muscle at 60 degrees C induced the liberation of actin from myofibrils. When a mixture of a myofibrillar fraction and a low-molecular sarcoplasmic fraction prepared from squid muscle was heated at 60 degrees C, actin liberation occurred. When a myofibrillar fraction was heated with ATP, AMP, or IMP, actin liberation occurred. Hence, AMP is perhaps one of the factors causing actin liberation in postmortem squid muscle. It was found that AMP and IMP reversibly dissociated actomyosin of chicken, bovine, and porcine skeletal muscles into actin and myosin on incubation at 0 degrees C at pH 7.2 in 0.2 M KCl. These results led us to conclude that AMP and IMP were the most responsible factors causing actin liberation from myofibrils in the heated muscle and causing reversible dissociation of actomyosin on storage of skeletal muscle at a low temperature. Hence, AMP and IMP are possible factors causing the resolution of rigor mortis in muscles.     

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.     

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.     

A simple method for the isolation of actin from myxomycete plasmodia.

A new, simple method for the isolation of actin from myxomycete plasmodia has been developed. Plasmodium myosin B was incubated at 55 degrees C for 15 min in the presence of ATP or was treated with 90% acetone. By this treatment myosin was denatured completely. Actin was then extracted with a dilute ATP and cysteine solution from the heat- or acetone-treated myosin B. The method is simple and almost pure actin was obtained in high yield. The purified G-actin polymerized to F-actin on addition of 0.1 M KCl or 2 mM MgCl2. The viscosity of the purified F-actin was 8-10 dl/g. The F-actin activated muscle myosin ATPase, and actomyosin synthesized from the F-actin and muscle myosin showed superprecipitation on addition of ATP.     

Characteristics of microsome-bound lactate dehydrogenase from skate white muscles]

Microsomes possessing the lactate dehydrogenase (LDH) activity were isolated from white driving muscles of the skate (Raja clavata) using differential centrifugation. It was shown that the increase of the ionic strength after addition of 0.6 M KCl and alkalinization of the medium result in the solubilization of the LDH activity - by 50% and 80%, respectively. The Km values for pyruvate and NADH are 171 microM and 7.5 mM, respectively. Membrane-bound LDH, is not inhibited by pyruvate excess (up to 20 mM); the rate of the enzyme inactivation by trypsin is 3 times as low as that of the solubilized enzyme. The existence of two-membrane-bound LDH pools is postulated. The enzyme from the first pool is bound to the membrane by electrostatic whereas the second pool LDH - by hydrophobic forces.     

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.     

Dinitrophenylation of rabbit skeletal actomyosin.

Dinitrophenylated reconstituted or natural actomyosin effected changes in the Ca2+ sensitivity which were dependent upon the ionic strength of the reaction medium. Dinitrophenylation of reconstituted actomyosin in 0.6 M KCl led to the incorporation of 2-6 mol of the reagent per 5-10(5) g of protein and it possessed considerable Ca2+ sensitivity. Dinitrophenylated natural actomyosin under the same conditions lost most of its Ca2+ sensitivity when 1.3-5.4 mol of the dinitrophenyl group were bound. The myosin from these modified actomyosins did not lose Ca2+ sensitivity and the myosin was labeled only with 0.4-1.7 mol of the dinitrophenyl group. Dinitrophenylation of both kinds of actomyosin in 0.06 M KCl abolished the Ca2+ sensitivity; the myosin from the modified actomyosins also lost Ca2+ sensitivity. Myosin alone was more susceptible to a loss of Ca2+ sensitivity than myosin in actomyosin. Actin protected the ability of myosin to sense Ca2+ regulated actin in modified actomyosin at 0.6 M KCl but not at 0.06 M KCl. Actomyosin dinitrophenylated in the presence of ATP lost Ca2+ sensitivity. However, the myosin from this actomyosin possessed Ca2+ sensitivity. Thiolysis of the dinitrophenylated actomyosin by 2-mercaptoethanol at low ionic strength did not restore the Ca2+ sensitivity of this actomyosin or its myosin although there was a significant loss of the dinitrophenyl group.     

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.     

Interactions between actin, myosin, and an actin-binding protein from rabbit alveolar macrophages. Alveolar macrophage myosin Mg-2+-adenosine triphosphatase requires a cofactor for activation by actin.

The interactions were analyzed between actin, myosin, and a recently discovered high molecular weight actin-binding protein (Hartwig, J. H., and Stossel, T. P. (1975) J. Biol Chem.250,5696-5705) of rabbit alveolar macrophages. Purified rabbit alveolar macrophage or rabbit skeletal muscle F-actins did not activate the Mg2+ATPase activity of purified rabbit alveolar macrophage myosin unless an additional cofactor, partially purified from macrophage extracts, was added. The Mg2+ATPase activity of cofactor-activated macrophage actomyosin was as high as 0.6 mumol of Pi/mg of myosin protein/min at 37 degrees. The macrophage cofactor increased the Mg2+ATPase activity of rabbit skeletal muscle actomyosin, and calcium regulated the Mg2+ATPase activity of cofactor-activited muscle actomyosin in the presence of muscle troponins and tropomyosin. However, the Mg2+ATPase activity of macrophage actomyosin in the presence of the cofactor was inhibited by muscle control proteins, both in the presence and absence of calcium. The Mg2+ATPase activity of the macrophage actomyosin plus cofactor, whether assembled from purified components or studied in a complex collected from crude macrophage extracts, was not influenced by the presence of absence of calcium ions. Therefore, as described for Acanthamoeba castellanii myosin (Pollard, T. D., and Korn, E. D. (1973) J. Biol. Chem. 248, 4691-4697), rabbit alveolar macrophage myosin requires a cofactor for activation of its Mg2+ATPase activity by F-actin; and no evidence was found for participation of calcium ions in the regulation of this activity.In macrophage extracts containing 0.34 M sucrose, 0.5 mM ATP, and 0.05 M KCl at pH 7.0,the actin-binding protein bound F-actin into bundles with interconnecting bridges. Purified macrophage actin-binding protein in 0.1 M KCl at pH 7.0 also bound purified macrophage F-actin into filament bundles. Macrophage myosin bound to F-actin in the absence but not the presence of Mg2+ATP, but the actin-binding protein did not bind to macrophage myosin in either the presence or absence of Mg2+ATP.     

Free and membrane-bound lactate dehydrogenase from white driving muscles of skate.

Purified cytoplasmic and membrane-bound lactate dehydrogenases (LDH) from white muscle of skate were characterized, Km for pyruvate and NADH for purified LDH were 150 +/- 16 and 29 +/- 7 microM, and for membrane-bound LDH were 185 +/- 22 and 7.5 +/- 1.5 microM, respectively. The membrane-bound enzyme was not inhibited by high pyruvate concentration (up to 20 mM) in contrast to purified LDH. Part of membrane-bound LDH was released by incubation in solutions with a high level of KCl (up to 1 M) or at alkaline pH. The inactivation rate during trypsin digestion for solubilized LDH was 2-3-fold higher than that for the membrane-bound enzyme.     

Thin filaments of molluscan catch muscles contain a calponin-like protein

A novel 40 kDa protein was detected in native thin filaments from catch muscles of the mussel Crenomytilus grayanus. The MALDY-TOF analysis of the protein showed a 40% homology with the calponin-like protein from the muscle of Mytilus galloprovincialis (45 kDa), which has a 36% homology with smooth muscle calponin from chicken gizzard (34 kDa). The amount of the calponin-like protein in thin filaments depends on isolation conditions and varies from the complete absence to the presence in amounts comparable with that of tropomyosin. The most significant factor that determines the contact of the protein in thin filaments is the temperature of solution in which thin filaments are sedimented by ultracentrifugation during isolation. At 22 degrees C and optimal values of both pH and ionic strength of the extraction solution, total calponin-like protein coprecipitates with thin filaments. At 2 degrees C it remains in the supernatant. The 40 kDa calponin-like protein from the mussel Crenomytilus grayanus has similar properties with smooth muscle calponin (34 kDa). It is thermostable and inhibits the actin-activated Mg -ATPase activity of actomyosin. In addition, the 40 kDa calponin-like protein isolated without using thermal treatment contains endogenous kinases. It was found that the calponin-like protein can be phosphorylated by endogenous kinases in the Ca -independent manner. These results indicate that the calponin-like protein from the catch muscle of the mussel Crenomytilus grayanus is a new member of the calponin family. The role of proteins from this family both in muscle and ponmuscle cells is still obscure. We suggest that the calponin-like protein is involved in the Ca -independent regulation of smooth muscle contraction.     

Changes in hexokinase activity during muscle alteration

Changes in extractability and activity of hexokinase (HK) were studied under the action of heating and of urea on skeletal muscles of Rana temporaria L., and besides the stability of this enzyme in muscle extract to those agents in vitro was examined. Under a 15 minutes heating of muscle, a decrease in extractability (the activity calculated for 1 g of tissue) and activity (the activity calculated for 1 mg of protein) of hexokinase is first revealed at 37 degrees C. Then the enzyme extractability decreases gradually in accordance to the decrease in extractability of the total water-soluble protein; the level of hexokinase activity attained at 37 degrees does not change up to 40 degrees. At 42 degrees the activity of the enzyme is completely inhibited. Under the heating of the muscle extract, the decrease of enzyme activity takes place at 36 degrees, the level achieved being stable up to 42 degrees C. Under the action of urea on the muscle at the reversible phase of alteration (1 M urea from 5 minutes to 2 hours at room temperature, 1 M urea for 9 hours at + 4 degrees C), hexokinase activity increases, calculated for 1 g of tissue and for 1 mg of protein. Under the irreversible disappearance of muscle excitability (1 M urea during 9 hours, 2 M urea during 2 hours at room temperature) no hexokinase activity was revealed. The activation of the enzyme is discussed in connection with the data on the increase of ATP content in muscle under the urea alteration. The treatment of the enzyme in muscle extract with 1 M urea decreases its activity in 30 minutes down to 67%; the level achieved does not change during 20 hours.     

Changes in lactate dehydrogenase and glucose-6-phosphate dehydrogenase activity in the course of muscle alteration]

Changes of outflux, extractability and activity of lactate dehydrogenase (LDG) and glucose-6-phosphate dehydrogenase (G-6PhDG) of muscles under the action of heating (at 32--44 degrees for 15 min) and urea (1 M during 10 min, 30 min, 2 hr. and 9 hr.) on the skeletal muscles of R. temporaria L. were studied. Under the thermal action not accompanied by contracture and fall of the excitability (32--36 degrees), the increase of outflux of LDG out of muscles into surrounding solutions is observed. G-6-PhDG in the external medium under any heating action was not revealed. Extractibility of LDG and G-6-PhDG did not change. Under the thermal action accompanied by the fall of excitability and by the contracture, along with the prolong increase of outflux of LDG, a decrease of extractability of LDG takes place. The decrease of G-6-PhDG is set at 42 degrees. Under the alteration of muscles by urea in the period of the temporary fall of excitability and contracture (10 and 30 min) an increase of the outflux of LDG out of muscles is observed. G-6PhDG in the surrounding medium was not revealed up to 9 hr. of incubation of muscle. In the period of the recovery of the excitability and relaxation of muscles (2hr.) the outflux of LDG approaches the control level. During the temporary loss and recovery of excitability, the extractability of LDG and G-6-PhDG does not change. In the period of irreversible contracture and loss of the excitability (6--10 hr.) a sharp increase of outflux of LDG out of muscles takes place. The extractability of the examined enzymes, especially of LDG, decreases.     

The inactivation by concanavalin A of the ecto-ATPase and ectoacetylcholinesterase of intact skeletal muscles

The (Ca2+ or Mg2+)-activated ectophosphohydrolase of intact frog muscle liberates, in situ, about 37 mumol inorganic phosphate/g muscle in 20 min at 20 degrees C with 10 mM ATP. Pretreatment with concanavalin A (ConA) at 4 degrees C for 18 h caused ectoenzyme inactivation which plateaued at 35-40% of the control rate. The inhibition was concentration dependent, being maximal at about 500 micrograms ConA/mL Ringer's solution. The lectin mediated its effect via the membrane glycoproteins since the inhibition was specifically prevented by alpha-methyl D-mannopyranoside. As the temperature increased from 10 to 40 degrees C, the ectoenzyme activity of untreated muscles increased linearly between 10 and 35 degrees C, with a "break point" and a clear change in slope at 35 degrees C. When treated with ConA the activity increased linearly from 10 to 40 degrees C, eliminating the transition temperature. The findings suggested that a phase transition toward fluidity in the lipid bilayer may have occurred at 35 degrees C and that this was abolished by the lectin binding. Hence we perturbed the surface membrane phospholipids of muscle pretreated with the lectin. Phospholipase C increased the activation by the lectin; phospholipase D had no effect, but phospholipase A2 completely prevented it. The lectin may require the more fluid fatty acyl chains of membrane lipids to achieve inhibition of this ecto-ATPase. Ectoacetylcholinesterase, in situ, and its inactivation by ConA were measured directly on whole, intact skeletal muscles.     

Interactions of muscle beta-connectin with myosin, actin, and actomyosin at low ionic strengths

The interaction of the muscle elastic protein connectin with myosin and actin filaments was investigated by turbidimetry, viscosity, flow birefringence measurements, and electron microscopic observations. In KCl concentrations lower than 0.15 M at pH 7.0 at 25 degrees C, both myosin and actin filaments were aggregated by connectin. Myosin filaments were entangled with each other in the presence of connectin. Actin filaments were assembled into bundles under the influence of connectin just as under that of alpha-actinin. The physiological significance of the interactions of connectin with myosin and actin filaments is discussed in relation to the localization of connectin in myofibrils. The Mg2+-activated ATPase activity of actomyosin was appreciably enhanced by connectin in the presence of KCl concentrations lower than 0.1 M. The extent of activation by connectin was smaller than by alpha-actinin. The enhancement of the ATPase activity may be due to acceleration of the onset of superprecipitation of actomyosin.     

Lactate and malate dehydrogenases in the muscles and male genital tract of the rabbit.

Average lactate dehydrogenase (LDH) isoenzyme patterns the content of H subunits, total LDH activity, total malate dehydrogenase (MDH) activity and the m- MDH/s-MDH ratio were determined in twelve muscles and the male genital tract of the rabbit. LDH(1) was the predominant form in the heart, soleus and masseter muscles, LDH(3) in the lingual muscles and LDH(5) in the other muscles analysed. In the muscles, an increase in the percentual proportion of M subunits was accompanied, by a proportional increase in total LDH activity and a decrease in total MDH activity, especially m-MDH. LDH isoenzyme patterns and LDH and MDH activities are useful for obtaining some idea about the proportion of individual muscle fibres. Activity accounted for by H subunits was roughly the same in all the muscles analysed, indicating that the synthesis of H subunits is independent of the type of muscle fibre and of the oxygen supply of the muscular tissue, and also that isoenzymes composed chiefly of H subunits are not localized preferentially in the mitochondria. Similar relationships between LDH isoenzymes and LDH and MDH activities were found in the testicular and epididymal tissues. The tests and the head of the epididymis mainly contain LDH isoenzymes composed of H subunits. The total LDH activity in these tissues is relatively low and their MDH activity is relatively high compared with the body and tail of the epididymis. The proportion of H subunits in the ampulla, the seminal vesicles, the coagulating glands and the prostate is also high. Cowper's glands have a high LDH(5) and LDH(4) concentration. One of two LDHx isoenzymes were found in the testes and spermatozoa.