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.     

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.     

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.     

The activities of some enzymes concerned with energy metabolism in mammalian muscles of differing pigmentation

1. Extractable hexokinase activity was measured in the red and white skeletal muscles of the rabbit and in the hearts and diaphragms of four animal species differing markedly in size. Activities vary over a 40-fold range, being least in white skeletal muscle of the laboratory rabbit and greatest in mouse heart. 2. Hexokinase activities correlate approximately with capacities to undertake reactions of the tricarboxylic acid cycle as determined by succinate oxidase assays. Both enzyme activities seem best related to the average contractile-energy expenditure per unit weight of muscle over an extended period, rather than to the rapidity of individual contractions. 3. Hexokinase and succinate oxidase activities cannot be related to a muscle's content of soluble pigment. They display an inverse relationship with activities of phosphorylase and glycolytic enzymes, but only within the group of rabbit skeletal muscles whose oxidative capacities are at the lower end of the observed range. 4. Total glycogen-UDP glucosyltransferase activities do not vary significantly between rabbit skeletal muscles, although those of hexokinase differ by about sixfold. On the average, glucose 6-phosphate is probably oxidized directly. However, observations cited in the literature suggest that muscles with an active hexokinase may well preferentially accumulate glycogen when glucose is present in excess of the fibres' capacity to oxidize it. 5. When considered with published results obtained in vivo, the present findings indicate that phosphorylase has a minor role in the energy expenditure of muscles with a predominantly oxidative metabolism. In these, the major substrates appear to be blood glucose, fatty acids and possibly lipids. 6. The histochemical criteria by which muscle fibres are commonly described as red or white are inadequate.     

Actin Degradation in the Metamorphosing Bullfrog Tadpole Tail

Degradation of tail muscle proteins was investigated during metamorphosis of Rana catesbeiana , tadpole. Regressing tail muscle contained actomyosin which was comparable to that of non-regressing tail muscle in its physico-chemical character, althouth the actomyosin content of the former tissue decreased as compared to the latter. However, when muscle proteins were extracted in the SDS-containing medium (TSM) and analyzed by SDS-polyacrylamide gel electrophoresis, we found that the protein band corresponding to actin disappeared completely during the late climax stage of metamorphosis. Detailed studies on this phenomenon showed that the apparent absence of actin on SDS-polyacrylamide gel electrophoresis was dependent upon the metamorphic stages of the tadpoles investigated. When TSM extract from the premetamorphic tadpole tail muscles which contained actin was incubated with the same extract from tadpoles of the climax stage, actin derived from premetamorphic tadpole disappeared on gel electrophoresis, indicating that tail muscle tissues of the climax stages contain the actin-degrading enzyme. Characterization of the enzyme was performed with a crude extract using actin prepared from rabbit thigh muscle as a substrate. Actin degrading activity showed incubation time- and temperature-dependency and the activity decreased gradually when the extract was preheated at increasing temperatures with the complete inactivation at 100°C. The major degradation products of actin hydrolysis by the enzyme had a Mr=28,000 and 14,000 which indicated the enzyme splits actin at a specific point. The activity had an optimum pH of 7.5 and was inhibited by leupeptin and iodoacetate and required the presence of a thiol reagent.     

Ascaris suum hexokinase: purification and possible function in compartmentation of glucose 6-phosphate in muscle.

Hexokinase (EC 2.7.1.1) is present in a soluble and a bound form in homogenates of Ascaris suum muscle. Cellulose acetate electrophoresis, isoelectric focusing, and ion exchange chromatography confirmed the presence of only one molecular form of hexokinase in this muscle. A procedure for purifying hexokinase from Ascaris muscle has been developed utilizing ion-exchange chromatography, ammonium sulfate fractionation and gel filtration. The enzyme is a monomer with a molecular weight of 100 000 as determined by sodium dodecyl sulfate gel filtration. The Stokes' radius, diffusion coefficient, and frictional ratio have been determined. The apparent Michaelis constants for glucose and ATP are 4.7-10(-3) M and 2.2-10(-4) M, respectively. Ascaris hexokinase also exhibits end-product inhibition by glucose 6-phosphate and ADP. It is postulated that the kinetic parameters of the enzyme are the results of its function, that of generating glucose 6-phosphate primarily for glycogen synthesis.     

Activity, hexokinase isoenzyme spectrum and various factors of their regulation in liver and skeletal muscles of young piglets]

Hexokinase in the liver of 1- and 5-day-old piglets is presented by four isoforms and in the skeletal muscles--by two ones. The enzyme activity in the liver and skeletal muscles of 5-day-old piglets is much higher than in 1-day-old ones. The increased hexokinase activity in the tissues of piglets during the first days of life appears to be due to the changes in their isoenzyme spectrum. The hexokinase activity and isoenzyme spectrum in the investigated tissue were affected by insulin, cortisol and 24 hours long starvation. These changes depended upon the age of the animals and differed in various organs and tissues: in 1-day-old piglets they were more pronounced in the skeletal muscles, while in 5-days-old animals--in the liver.     

Increased expression of GLUT-4 and hexokinase in rat epitrochlearis muscles exposed to AICAR in vitro.

Exercise acutely stimulates muscle glucose transport and also brings about an adaptive increase in the capacity of muscle for glucose uptake by inducing increases in GLUT-4 and hexokinase.(1) Recent studies have provided evidence that activation of AMP protein kinase (AMPK) is involved in the stimulation of glucose transport by exercise. The purpose of this study was to determine whether activation of AMPK is also involved in mediating the adaptive increases in GLUT-4 and hexokinase. To this end, we examined the effect of incubating rat epitrochlearis muscles in culture medium for 18 h in the presence or absence of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), which enters cells and is converted to the AMP analog ZMP, thus activating AMPK. Exposure of muscles to 0.5 mM AICAR in vitro for 18 h resulted in an approximately 50% increase in GLUT-4 protein and an approximately 80% increase in hexokinase. This finding provides strong evidence in support of the hypothesis that the activation of AMPK that occurs in muscle during exercise is involved in mediating the adaptive increases in GLUT-4 and hexokinase.     

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.     

Chronic activation of 5'-AMP-activated protein kinase increases GLUT-4, hexokinase, and glycogen in muscle.

This study was designed to determine whether chronic chemical activation of AMP-activated protein kinase (AMPK) would increase glucose transporter GLUT-4 and hexokinase in muscles similarly to periodic elevation of AMPK that accompanies endurance exercise training. The adenosine analog, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), has previously been shown to be taken up by cells and phosphorylated to form a compound (5-aminoimidazole-4-carboxamide ribonucleotide) that mimics the effect of AMP on AMPK. A single injection of AICAR resulted in a marked increase in AMPK in epitrochlearis and gastrocnemius/plantaris muscles 60 min later. When rats were injected with AICAR (1 mg/g body wt) for 5 days in succession and were killed 1 day after the last injection, GLUT-4 was increased by 100% in epitrochlearis muscle and by 60% in gastrocnemius muscle in response to AICAR. Hexokinase was also increased approximately 2. 5-fold in the gastrocnemius/plantaris. Gastrocnemius glycogen content was twofold higher in AICAR-treated rats than in controls. Chronic chemical activation of AMPK, therefore, results in increases in GLUT-4 protein, hexokinase activity, and glycogen, similarly to those induced by endurance training.     

The activities of phosphorylase, hexokinase, phosphofructokinase, lactate dehydrogenase and the glycerol 3-phosphate dehydrogenase in muscles from vertebrates and invertebrates

1. The maximum activities of hexokinase, phosphorylase and phosphofructokinase have been measured in extracts from a variety of muscles and they have been used to estimate the maximum rates of operation of glycolysis in muscle. These estimated rates of glycolysis are compared with those calculated for the intact muscle from such information as oxygen uptake, glycogen degradation and lactate formation. Reasonable agreement between these determinations is observed, and this suggests that such enzyme activity measurements may provide a useful method for comparative investigations into quantitative aspects of maximum glycolytic flux in muscle. 2. The enzyme activities from insect flight muscle confirm and extend much of the earlier work and indicate the type of fuel that can support insect flight. The maximum activity of hexokinase in some insect flight muscles is about tenfold higher than that in vertebrate muscles. The activity of phosphorylase is greater, in general, in vertebrate muscle (particularly white muscle) than in insect flight muscle. This is probably related to the role of glycogen breakdown in vertebrate muscle (particularly white muscle) for the provision of ATP from anaerobic glycolysis and not from complete oxidation of the glucose residues. The activity of hexokinase was found to be higher in red than in white vertebrate muscle, thus confirming and extending earlier reports. 3. The maximum activity of the mitochondrial glycerophosphate dehydrogenase was always much lower than that of the cytoplasmic enzyme, indicating that the former enzyme is rate-limiting for the glycerol 3-phosphate cycle. From the maximum activity of the mitochondrial enzyme it can be calculated that the operation of this cycle would account for the reoxidation of all the glycolytically produced NADH in insect flight muscle but it could account for only a small amount in vertebrate muscle. Other mechanisms for this NADH reoxidation in vertebrate muscle are discussed briefly.     

Changes in the activity of various enzymes of carbohydrate metabolism in the liver and skeletal muscles of pigs during ontogenesis

Hexokinase, glucokinase, phosphofructokinase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activity was studied in the liver and musculus quadriceps femoris of 110-day foetuses 1, 2, 3, 30 and 60-day piglets and in adult pigs. The activity of all enzymes in the tissues of newborn piglets is considerably higher than in the tissues of foetuses. The activity of hexokinase in both tissues of piglets increases in the first days after birth and lowers by the one month age. The phosphofructokinase activity in the skeletal muscles and the glucokinase one in the pig liver increase during the postnatal development. The activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in both tissues of pigs increases after birth and then decreases. Glucose metabolism in the pig liver at all stages of odontogenesis proceeds more intensively by the pentose phosphate pathway, and in the skeletal muscles--by glycolytic one.     

Myosin and actomyosin from human skeletal muscle.

Human skeletal natural actomyosin contained actin, tropomyosin, troponin and myosin components as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Purified human myosin contained at least three light chains having molecular weights (+/-2000) of 25 000, 18 000 and 15 000. Inhibitory and calcium binding components of troponin were identified in an actin-tropomyosin-troponin complex extracted from acetone-dried muscle powder at 37 degrees C. Activation of the Mg-ATPase activity of Ca2+-sensitive human natural or reconstituted actomyosin was half maximal at approximately 3.4 muM Ca2+ concentration (CaEGTA binding constant equals 4.4 - 10(5) at pH 6.8). Subfragment 1, isolated from the human heavy meromyosin by digestion with papain, appeared as a single peak after DEAE-cellulose chromatography. In the pH 6-9 range, the Ca2+-ATPase activity of the subfragment 1 was 1.8- and 4-fold higher that the original heavy meromyosin and myosin, respectively. The ATPase activities of human myosin and its fragments were 6-10 fold lower than those of corresponding proteins from rabbit fast skeletal muscle. Human myosin lost approximately 60% of the Ca2+-ATPase activity at pH 9 without a concomitant change in the number of distribution of its light chains. These findings indicate that human skeletal muscle myosin resembles other slow and fast mammalian muscles. Regulation of human skeletal actomyosin by Ca2+ is similar to that of rabbit fast or slow muscle.     

Glucose metabolism in the mucosa of the small intestine. The effect of glucose on hexokinase activity

1. The effect of three dietary components on hexokinase activity in the mucosa of rat small intestine was studied in vivo. Glucose, amino acids or an emulsion of monoglyceride with long-chain fatty acids were given by stomach tube to previously starved rats, and hexokinase activity was determined in the particle-free supernatant of mucosal homogenates. The formation of lactate from glucose and glucose 6-phosphate respectively was also measured. 2. When the three dietary components were given in isocaloric amounts, only glucose brought about an increase in hexokinase activity. 3. Intravenous injection of a similar amount of glucose to that given orally did not alter hexokinase activity. 4. An increase in the hexokinase activity of the particle-free supernatant prepared from mucosal homogenates was also observed after sacs of the small intestine of starved rats had been incubated in vitro in a medium containing glucose. Hexokinase activity increased to the values observed in corresponding preparations from fed rats, and this increase was strictly glucose-dependent.     

Adaptation in synergistic muscles to soleus and plantaris muscle removal in the rat hindlimb.

Although the soleus muscle comprises only 6% of the ankle plantar flexor mass in the rat, a major role in stance and walking has been ascribed to it. The purpose of this study was to determine if removal of the soleus muscle would result in adaptations in the remaining gastrocnemius and plantaris muscles due to the new demands for force production imposed on them during stance or walking. A second purpose was to determine whether the mass or the fiber type of the muscle(s) removed was a more important determinant of compensatory adaptations. Male Sprague-Dawley rats underwent bilateral removal of soleus muscle, plantaris muscle, or both muscles. For comparison, compensatory hypertrophy was induced in soleus and plantaris muscles by gastrocnemius muscle ablation. After forty days, synergist muscles remaining intact were removed. Mass, and oxidative, glycolytic, and contractile enzyme activities were determined. Despite its role in stance and slow walking, removal of the soleus muscle did not elicit a measurable alteration in muscle mass, or in citrate synthase, lactate dehydrogenase, or myofibrillar ATPase activity in gastrocnemius or plantaris muscles. Similarly, removal of the plantaris muscle, or soleus and plantaris muscles, had no effect on the gastrocnemius muscle, suggesting that this muscle was able to easily meet the new demands placed on it. These results suggest that amount of muscle mass removed, rather than fiber type, is the most important stimulus for compensatory hypertrophy. They also suggest that slow-twitch motor units in the gastrocnemius muscle play an important role during stance and locomotion in the intact animal.     

Change in the ATPase activity of glycerinated muscle fibers under the influence of urethane]

The influence of uretane on ATPase activity and contractility of frog glycerinated muscle fibers was studied. Parallel decrease of contractility and ATPase activity of glycerinated muscles takes place upon toxic uretane concentration (1.01 M). A lower uretane concentration (0.34 M) inducing considerable increase of conservation time contractive reaction maintains a higher ATPase activity than does the control one; ATPase activity of the actomyosin from these models is the same as the control one.     

Myosin and actomyosin from human skeletal muscle

Human skeletal natural actomyosin contained actin, tropomyosin, troponin and myosin components as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Purified human myosin contained at least three light chains having molecular weights (±2000) of 25 000, 18 000 and 15 000. Inhibitory and calcium binding components of troponin were identified in an actin-tropomyosin-troponin complex extracted from acetone-dried muscle powder at 37°C. Activation of the Mg-ATPase activity of Ca²⁺-sensitive human natural or reconstituted actomyosin was half maximal at approximately 3.4 μM Ca²⁺ concentration (CaEGTA binding constant = 4.4 · 10⁵ at pH 6.8). Subfragment 1, isolated from the human heavy meromyosin by digestion with papain, appeared as a single peak after DEAE-cellulose chromatography. In the pH 6–9 range, the Ca²⁺-ATPase activity of the subfragment 1 was 1.8-and 4-fold higher that the original heavy meromyosin and myosin, respectively. The ATPase activities of human myosin and its fragments were 6–10 fold lower than those of corresponding proteins from rabbit fast skeletal muscle. Human myosin lost approximately 60% of the Ca²⁺-ATPase activity at pH 9 without a concomitant change in the number of distribution of its light chains. These findings indicate that human skeletal muscle myosin resembles other slow and fast mammalian muscles. Regulation of human skeletal actomyosin by Ca²⁺ is similar to that of rabbit fast or slow muscle     

Myosin from molluscan abalone, Haliotis discus. Isolation and enzymatic properties.

Actomyosin was extracted from smooth muscle of molluscan abalone with 0.1 M PPit pH 6.4. Myosin was separated from the actomyosin by centrifugation at 100,000 X g in the presence of 5 mM ATP and 10 mM MgCl2. Myosin in the supernatant was further purified by gel filtration on a Sepharose 4B column. Paramyosin contamination of the actomyosin preparation interfered with the isolation of myosin and complete removal of actin and paramyosin from the myosin has not been accomplished. The myosin appeared to consist of a single f-chain and a single g-chain, as examined by SDS-disc electrophoresis in 8 or 13.7% acrylamide gel. The ATPase [EC 3.6.1.3] activity of this myosin in 0.5 M KCL at neutral pH and at 0 degrees was rather unstable and decreased by 10-20% per day. The effects of rho-chloromercuribenzoate and EDTA on the ATPase activity were similar to those observed with other smooth muscle myosin but the dependence upon pH or KCL concentration was different.     

Purification of a hexokinase-binding protein from the outer mitochondrial membrane.

Brain hexokinase (ATP:D-hexose-6-phosphotransferase, EC 2.7.1.1) binds selectively to the outer membrane of rat liver mitochondria but not to inner mitochondrial or microsomal membranes nor to the plasma membrane of human erythrocytes. A protein having subunit molecular weight of 31,000, determined by sodium dodecyl sulfate-gel electrophoresis, has been highly purified from the outer mitochondrial membrane by repetitive solubilization with octyl-beta-D-glucopyranoside followed by reconstitution into membranous vesicles when the detergent is removed by dialysis. When incorporated into lipid vesicles, the protein confers the ability to bind brain hexokinase in a Glc-6-P-sensitive manner as is seen with the intact outer mitochondrial membrane. Hexokinase binding ability and the 31,000 subunit molecular weight protein co-sediment during sucrose density gradient centrifugation. Both hexokinase binding ability and the 31,000 subunit molecular weight protein are resistant to protease treatment of the intact outer mitochondrial membrane while other membrane proteins are extensively degraded. It is concluded that this protein, designated the hexokinase-binding protein (HBP), is an integral membrane protein responsible for the selective binding of hexokinase by the outer mitochondrial membrane.