The activities of fructose 1,6-diphosphatase, phosphofructokinase and phosphoenolpyruvate carboxykinase in white muscle and red muscle

1. The activities of fructose 1,6-diphosphatase were measured in extracts of muscles of various physiological function, and compared with the activities of other enzymes including phosphofructokinase, phosphoenolpyruvate carboxykinase and the lactate-dehydrogenase isoenzymes. 2. The activity of phosphofructokinase greatly exceeded that of fructose diphosphatase in all muscles tested, and it is concluded that fructose diphosphatase could not play any significant role in the regulation of fructose 6-phosphate phosphorylation in muscle. 3. Fructose-diphosphatase activity was highest in white muscle and low in red muscle. No activity was detected in heart or a deep-red skeletal muscle, rabbit semitendinosus. 4. The lactate-dehydrogenase isoenzyme ratio (activities at high and low substrate concentration) was measured in various muscles because a low ratio is characteristic of muscles that are more dependent on glycolysis for their energy production. As the ratio decreased the activity of fructose diphosphatase increased, which suggests that highest fructose-diphosphatase activity is found in muscles that depend most on glycolysis. 5. There was a good correlation between the activities of fructose diphosphatase and phosphoenolpyruvate carboxykinase in white muscle, where the activities of these enzymes were similar to those of liver and kidney cortex. However, the activities of pyruvate carboxylase and glucose 6-phosphatase were very low in white muscle, thereby excluding the possibility of gluconeogenesis from pyruvate and lactate. 6. It is suggested that the presence of fructose diphosphatase and phosphoenolpyruvate carboxykinase in white muscle may be related to operation of the alpha-glycerophosphate-dihydroxyacetone phosphate and malate-oxaloacetate cycles in this tissue.     

Intestinal ammonia metabolism in ethanolic rats

Repeated treatment of ethanol for 5 consecutive days has produced significant biochemical changes in the small intestine of the rat. The LDH and SDH were inhibited leading to deficient energy output from the glycolytic pathway and the Krebs cycle. The changes in ammonia metabolic profiles of intestine and blood indicate the presence of hyperammonemia. Enzymatic reactions associated with urea production and the levels of urea were increased. GS activity decreased explaining low levels of glutamine. These studies implicate PNC in producing hyperammonemia during ethanol treatment.     

Enzymatic and mitochondrial responses to 5 months of aerial exposure in the slender lungfish Protopterus dolloi

Mitochondrial respiration and activities of key metabolic enzymes from liver and white skeletal muscle were compared between control aquatic slender lungfish Protopterus dolloi , and those exposed to air for 5 months. Activities of citrate synthase, glycogen phosphorylase, phosphofructokinase and pyruvate kinase in liver were not affected by air-exposure. In muscle, air-exposure reduced citrate synthase and pyruvate kinase activities (relative to tissue wet mass) by 63 and 50%, respectively. Liver carnitine palmitoyl transferase activity (relative to mitochondrial protein) decreased by half following air-exposure, but there was no change in muscle. In mitochondria isolated from muscle, state 3 and state 4 respiration were reduced by 74 and 89%, respectively following air-exposure, but liver mitochondria were not affected. In liver, air-exposure increased activities of ornithine-urea cycle enzymes including glutamine synthase, carbamoyl-phosphate synthase III and arginase, by 1·9- to 4·2-fold. Carbamoyl-phosphate synthase III activity could not be detected in muscle, indicating that urea is not synthesized in this tissue. These data suggest that skeletal muscle metabolism is downregulated in air-exposure, conserving energy and protein during a period when the animals cannot forage. In contrast, ATP production capacities in the liver are maintained, and this may permit expensive urea biosynthesis to continue during aerial exposure.     

Maximal activities of hexokinase, 6-phosphofructokinase,oxoglutarate dehydrogenase,and carnitine palmitoyltransferase in rat and avian muscles

The maximum activities of 6-phosphofructokinase and oxoglutarate dehydrogenase in muscle provide quantitative indices of the maximum capacities of anaerobic glycolysis and the Krebs cycle (i.e. the aerobic capacity) respectively. These activities were measured in red, white, and cardiac muscle of birds and the rat. The activities in the white pectoral muscle of the domestic fowl suggest that the Krebs cycle plus electron transfer could provide only about 1% of the rate of ATP production provided by anaerobic glycolysis whereas in pigeon pectoral muscle the predicted maximal rates from the two processes are similar. In contrast to domestic-fowl pectoral muscle, the white rat muscle, epitrochlearis, contains a significant activity of oxoglutarate dehydrogenase, which indicates that the Krebs cycle could provide about 12% of the maximum rate of ATP formation. This may be explained by a higher proportion of type-I and -IIA fibres in the rat muscle compared to the avian muscle. In the aerobic muscles of the rat the maximum activities of carnitine palmitoyl transferase indicate that fatty-acid oxidation could provide a high rate of ATP formation.     

白三叶和红三叶对人工融冻胁迫的生理响应差异

采用人工模拟融冻胁迫方法,通过测定白三叶(Trifolium repens)和红三叶(T.prat-ense)在融冻胁迫中叶片细胞膜透性、MDA含量、抗氧化酶(SOD、POD、CAT)活力、渗透调节物(脯氨酸、可溶性糖和蛋白质)含量变化,以揭示未来气候变化对三叶草的影响。结果表明,经历融冻胁迫循环后抗冻力强的白三叶植株能恢复生长,而抗冻力弱的红三叶枯萎死亡。在融冻阶段,两三叶草叶片细胞膜透性增大、抗氧化酶活力增高、MDA和渗透调节物含量大幅增加;在冻融阶段,两三叶草叶片细胞膜透性降低、MDA含量下降、抗氧化酶活力降低。但在融冻胁迫循环中,白三叶叶片POD和CAT活力高于红三叶,脯氨酸含量较红三叶高5倍,但细胞膜透性低于红三叶。白三叶在-5℃抗逆生理指标达到最大值,而红三叶在-10℃。白三叶对环境温度变化反应敏感,在-5℃通过快速激活抗氧化酶系统和积累渗透调节物以抑制膜脂过氧化和维护细胞水分平衡在融冻适应上起重要作用。白三叶具有较强的抗融冻能力,是未来值得应用推广的优良园林绿化植物。     

A comparative study of aldehyde dehydrogenase and alcohol dehydrogenase activities in crucian carp and three other vertebrates: apparent adaptations to ethanol production

Summary In the final step of the pathway producing ethanol in anoxic crucian carp (Carassius carassius L.), acetaldehyde is reduced to ethanol by alcohol dehydrogenase. The presence of aldehyde dehydrogenase in the tissues responsible for ethanol production could cause an undesired oxidation of acetaldehyde to acetate coupled with a reduction of NAD⁺ to NADH. Moreover, acetaldehyde could competitively inhibit the oxidation of reactive biogenic aldehydes. In the present study, the distribution of aldehyde dehydrogenase (measured with a biogenic aldehyde) and alcohol dehydrogenase (measured with acetaldehyde) were studied in organs of crucian carp, common carp (Cyprinus carpio L.), rainbow trout (Salmo gairdneri Richardson), and Norwegian rat (Rattus norvegicus Berkenhout). The results showed that alcohol dehydrogenase and aldehyde dehydrogenase activities were almost completely spatially separated in the crucian carp. These enzymes occurred together in the other three vertebrates. In the crucian carp, alcohol dehydrogenase was only found in red and white skeletal muscle, while these tissues contained exceptionally low aldehyde dehydrogenase activities. Moreover, the low aldehyde dehydrogenase activity found in crucian carp red muscle was about 1000 times less sensitive to inhibition by acetaldehyde than that found in other tissues and other species. The results are interpreted as demonstrating adaptations to avoid a depletion of ethanol production, and possibly inhibition of biogenic aldehyde metabolism.Abbreviations ADH alcohol dehydrogenase - ALDH aldehyde dehydrogenase - DOPAL 3,4-dihydroxyphenylacetaldehyde - MAO monoamine oxidase - PCA perchloric acid     

Some neurochemical consequences of repeated ethanol loading in rat brain

Neurochemical consequences of repeated ethanol treatment on energy and ammonia metabolism were studied in different regions of rat brain. Energy production was decreased as indicated by lowered lactate dehydrogenase and succinate dehydrogenase activities with possible lacticacidimia. Transamination of alanine and aspartate increased while the deamination of glutamate decreased in all the regions of brain. The deamination of AMP was slightly elevated in cerebral cortex and brain stem while it was inhibited in cerebellum. Ammonia levels were persistently high, despite stepped up glutamine synthesis and ureogenesis. The synergistic action of ammonia during ethanol intoxication is envisaged.     

Energy metabolism of carp swimming muscles

Summary Electromyography has been used to study the recruitment of red, pink and white muscle fibres of the Mirror carp at different swimming speeds. Locomotion below 0.3–0.5 L/S (lengths per second) is achieved primarily by fin movements after which the red myotomal muscle becomes active. Pink muscle fibres are the next type to be recruited at speeds around 1.1–1.5 L/S. White muscle is only used for fast cruising in excess of 2–2.5 L/S and during bursts of acceleration.Studies of the myofibrillar ATPase activities of these muscles have shown a ratio of 124 for the red, pink and white fibres respectively. The myosin low molecular weight components, which are characteristic of the myosin phenotype, have been investigated by SDS polyacrylamide electrophoresis. The light chain patterns of the pink and white muscles were identical and characteristic of the fast myosin phenotype. Red muscle myosin had a light chain pattern characteristic of slow muscles. It would appear that there is a relationship between the speed of contraction of the fibre types and the locomotory speed at which they are recruited.The activities of some enzymes of energy metabolism have also been determined in the three muscle types. Enzymes associated with oxidate metabolism have high, intermediate and low activities in the red, pink and white muscles respectively. Pyruvate kinase and lactate dehydrogenase activities were considerably higher in the pink than in either red or white muscles. It is suggested that the high capacity for anaerobic glycolysis of the pink muscle is associated with its recruitment for sustained effort at swimming speeds above which the fish can no longer meet all its energy requirements by gas exchange at the gills.Abbreviations used EDTA ethylenediamine tetraacetic acid - L/S lengths, sec^–1 - LDH Lactate dehydrogenase - PFK phosphofructokinase - SDS sodium dodecyl sulphate - TCA trichloroacetic acid     

Acute hyperammonemia activates branched-chain amino acid catabolism and decreases their extracellular concentrations: different sensitivity of red and white muscle

Hyperammonemia is considered to be the main cause of decreased levels of the branched-chain amino acids (BCAA), valine, leucine, and isoleucine, in liver cirrhosis. In this study we investigated whether the decrease in BCAA is caused by the direct effect of ammonia on BCAA metabolism and the effect of ammonia on BCAA and protein metabolism in different types of skeletal muscle. M. soleus (SOL, slow-twitch, red muscle) and m. extensor digitorum longus (EDL, fast-twitch, white muscle) of white rat were isolated and incubated in a medium with or without 500 μM ammonia. We measured the exchange of amino acids between the muscle and the medium, amino acid concentrations in the muscle, release of branched-chain keto acids (BCKA), leucine oxidation, total and myofibrillar proteolysis, and protein synthesis. Hyperammonemia inhibited the BCAA release (81% in SOL and 60% in EDL vs. controls), increased the release of BCKA (133% in SOL and 161% in EDL vs. controls) and glutamine (138% in SOL and 145% in EDL vs. controls), and increased the leucine oxidation in EDL (174% of controls). Ammonia also induced a significant increase in glutamine concentration in skeletal muscle. The effect of ammonia on intracellular BCAA concentration, protein synthesis and on total and myofibrillar proteolysis was insignificant. The data indicates that hyperammonemia directly affects the BCAA metabolism in skeletal muscle which results in decreased levels of BCAA in the extracellular fluid. The effect is associated with activated synthesis of glutamine, increased BCAA oxidation, decreased release of BCAA, and enhanced release of BCKA. These metabolic changes are not directly associated with marked changes in protein turnover. The effect of ammonia is more pronounced in muscles with high content of white fibres.     

Characteristics and Efficiency of Glutamine Production by Coupling of a Bacterial Glutamine Synthetase Reaction with the Alcoholic Fermentation System of Baker’s Yeast

Glutamine production with bacterial glutamine synthetase (GS) and the sugar-fermenting system of baker’s yeast for ATP regeneration was investigated by determining the product yield obtained with the energy source for ATP regeneration (i.e., glucose) for yeast fermentation. Fructose 1,6-bisphosphate was accumulated temporarily prior to the formation of glutamine in mixtures which consisted of dried yeast cells, GS, their substrate (glucose and glutamate and ammonia), inorganic phosphate, and cofactors. By an increase in the amounts of GS and inorganic phosphate, the amounts of glutamine formed increased to 19 to 54 g/liter, with a yield increase of 69 to 72% based on the energy source (glucose) for ATP regeneration. The analyses of sugar fermentation of the yeast in the glutamine-producing mixtures suggested that the apparent hydrolysis of ATP by a futile cycle(s) at the early stage of glycolysis in the yeast cells reduces the efficiency of ATP utilization. Inorganic phosphate inhibits phosphatase(s) and thus improves glutamine yield. However, the analyses of GS activity in the glutamine-producing mixtures suggested that the higher concentration of inorganic phosphate as well as the limited amount of ATP-ADP caused the low reactivity of GS in the glutamine-producing mixtures. A result suggestive of improved glutamine yield under the conditions with lower concentrations of inorganic phosphate was obtained by using a yeast mutant strain that had low assimilating ability for glycerol and ethanol. In the mutant, the activity of the enzymes involved in gluconeogenesis, especially fructose 1,6-bisphosphatase, was lower than that in the wild-type strain.     

beta-Glucuronidase activity in trained red and white skeletal muscle of mice.

We studied the effects of prolonged running exercise (5 days a week, 1.5 h per day at a speed of 17.6 m/min) on the activity of some acid hydrolases (beta-glucuronidase, beta-N-acetylglucosaminidase, acid phosphatase and cathepsin D) and three enzymes of energy metabolism (cytochrome c oxidase, lactate dehydrogenase and creatine kinase) in the distal and in the proximal, the predominantly white and red parts, respectively, of the vastus lateralis-muscle from mice. The acid hydrolase activity levels were 1.24--1.69 higher in untrained red muscle compared to untrained white muscle. The light training applied increased the activity of beta-glucuronidase in both red and white muscle. No other significant training effects were observed in the enzyme activities measured.     

The sleeper Bostrichthys sinensis (family Eleotridae) stores glutamine and reduces ammonia production during aerial exposure

Bostrichthys sinensis inhabits brackish water, living in the crevices of the river mouths of Shang Xi and Guangdong, China. In its natural habitat, it may encounter aerial exposure frequently during low tides, and it usually remains quiescent in the absence of water. Upon aerial exposure in the laboratory, the ammonia excretion rate decreased to one-fourth that of the submerged control. Although all the enzymes of the ornithine-urea cycle were detected in the liver of this fish, the activity of hepatic carbamoyl phosphate synthetase was too low for the cycle to be functioning. Indeed, ammonia accumulated in the tissues and was not converted to urea. Results indicate that ammonia produced through amino acid catabolism was detoxified to glutamine during the first 24 h of aerial exposure. The excess amount of glutamine stored in the muscle during this period couldaccount approximately for the reduction in ammonia equivalent excreted. There was indeed a significant increase in the activity of glutamine synthetase from the liver of specimens exposed to terrestrial conditions. In contrast to the production of alanine, formation of glutamine is energetically expensive. Since B. sinensis remained relatively inactive on land, the reduction in energy demand for muscular activity might provide it with the opportunity to exploit glutamine formation as a means to detoxify ammonia. After 72 h of aerial exposure, B. sinensis reduced internal ammonia production, possibly through reductions in proteolysis and amino acid catabolism, to avoid excessive accumulation of ammonia.     

力竭游泳对大鼠不同类型肌纤维自由基代谢和血清酶的影响

本研究观察了负重力竭游泳前后大鼠不同类型肌纤维中与自由基代谢有关的若干指标 ,如丙二醛 (MDA)、超氧化物歧化酶 (SOD)和血清肌酸激酶 (CK)、乳酸脱氢酶 (LDH)、谷草转氨酶 (GOT)活性的变化     

8-Oxoguanosine and uracil repair of nuclear and mitochondrial DNA in red and white skeletal muscle of exercise-trained old rats.

Oxoguanine DNA glycosylase (OGG1) and uracil DNA glycosylase (UDG) are two of the most important repair enzymes that are involved in the base excision repair processes to eliminate oxidative damage from mammalian DNA, which accumulates with aging. Red and white skeletal muscle fibers have very different antioxidant enzyme activities and resistance to oxidative stress. In this paper, we demonstrate that the activity of OGG1 is significantly higher in the red type of skeletal muscle compared with white fibers from old rats. Exercise training resulted in increased OGG1 activity in the nuclei of red fibers and decreased activity in nuclei of white fibers and in the mitochondria of both red and white fibers. The activities of UDG were similar in both red and white muscle fibers. Exercise training appears to increase the activity of UDG in the nuclei and mitochondria. However, exercise training affects the activity of OGG1 in nuclei and mitochondria differently, suggesting different regulation of the enzymes. In contrast, UDG showed similar activities in nuclei and mitochondrial extracts of exercise-trained animals. These data provide evidence for differential regulation of UDG and OGG1 in maintaining fidelity of DNA in oxidatively stressed cells.     

Influence of ribose on adenine salvage after intense muscle contractions.

The influence of ribose supplementation on skeletal muscle adenine salvage rates during recovery from intense contractions and subsequent muscle performance was evaluated using an adult rat perfused hindquarter preparation. Three minutes of tetanic contractions (60 tetani/min) decreased ATP content in the calf muscles by approximately 50% and produced an equimolar increase in IMP. Effective recovery of muscle ATP 1 h after contractions was due to reamination of IMP via the purine nucleotide cycle and was complete in the red gastrocnemius but incomplete in the white gastrocnemius muscle section. Adenine salvage rates in recovering muscle averaged 45 +/- 4, 49 +/- 5, and 30 +/- 3 nmol. h(-1). g(-1) for plantaris, red gastrocnemius, and white gastrocnemius muscle, respectively, which were not different from values in corresponding nonstimulated muscle sections. Adenine salvage rates increased five- to sevenfold by perfusion with approximately 4 mM ribose (212 +/- 17, 192 +/- 9, and 215 +/- 14 nmol. h(-1). g(-1) in resting muscle sections, respectively). These high rates were sustained in recovering muscle, except for a small (approximately 20%) but significant (P < 0.001) decrease in the white gastrocnemius muscle. Ribose supplementation did not affect subsequent muscle force production after 60 min of recovery. These data indicate that adenine salvage rates were essentially unaltered during recovery from intense contractions.     

Is digestive capacity limiting growth at low temperatures in roach?

In roach Rutilus rutilus growth ceases below a temperature threshold of 12° C. This cessation of growth is accompanied by a reduction in feeding. Do roach decrease feeding in the cold because of reduced energy demand, caused by the decelerating effect of low temperature on metabolism and growth, or is feeding directly limited by low temperatures, leading to reduced growth rates? It was found that at low temperatures the intake and digestion of food may be limited by reduced activities of digestive enzymes. Trypsin, amylase and γ‐glutamyl transferase showed a negative compensation with respect to temperature, resulting in very low activities at acclimation temperatures of ≤12° C. Trypsin activity, falling from 400·5 ± 131·2 U g^?1 fresh mass of the gut at 27° C to 12·5 U g^?1 fresh mass at 4° C, displayed the strongest linear correlation with growth rates, suggesting that trypsin activities may set a limit to growth in the low temperature range. If protein digestion is limiting at low temperatures, this should be reflected in reduced concentrations of amino acid in the white muscle. The size of the total amino acid pool was not affected by temperature acclimation and ranged between 19·2 ± 6·2 and 25·2 ± 3·6 µmol g^?1 fresh mass of the white muscle. A decrease, however, was found of several amino acids, mainly of threonine and glutamine, in the low temperature range. Low concentrations of the essential amino acid threonine (0·14 ± 0·03 µmol g^?1 fresh mass at 12° C and 0·12 ± 0·05 µmol g^?1 fresh mass at 4° C) were probably due to nutritional or digestional limitations and may therefore have resulted from reduced trypsin activity in the cold. The non‐essential amino acid glutamine, however, can be endogenously synthesized and its low level observed at 4° C (0·16 ± 0·09 µmol g^?1 fresh mass) was not necessarily a result of low trypsin activities. It is more likely that low temperatures impair glutamine synthesis. The possibility that glutamine concentrations may be down regulated under conditions when anabolic processes are not advantageous is discussed.     

Mutually exclusive muscle designs: the power output of the locomotory and sonic muscles of the oyster toadfish (Opsanus tau)

Animals perform a vast array of motor activities. Although it has generally been accepted that muscles are well suited to the function that they must perform, specialization for performing one function may compromise their ability for carrying out another. We examined this principle in the toadfish muscular system: slow-twitch red and fast-twitch white myotomal muscles are used for powering swimming at relatively low frequencies, while the superfast swimbladder muscle powers mating calls by contracting at 100 Hz. We measured muscle power output over a wide range of frequencies. The red and white locomotory muscles could not generate power over ca. 2.2 and 12 Hz, respectively and, hence, could not power sound production. In contrast, the swimbladder muscle has many specializations that permit it to generate power at frequencies in excess of 100 Hz. However, these specializations drastically reduce its power output at low frequencies: the swimbladder muscle generated only one-twentieth of the power of the red muscle and one-seventh of the power of the white muscle at the frequencies used during swimming. To generate the same total power needed for swimming would require unfeasibly large amounts of swimbladder muscle that could not fit into the fish. Hence, the designs of the swimbladder and locomotory muscles are mutually exclusive.     

The effect of endurance-training on the maximum activities of hexokinase, 6-phosphofructokinase,citrate synthase,and oxoglutarate dehydrogenase in red and white muscles of the rat

Adult female rats were subjected to an eleven-week endurance-training programme, and, for the first time, the maximum activities of enzymes that can indicate the quantitative capacities of both anaerobic glycolysis and the Krebs cycle in muscle (viz. 6-phosphofructokinase and oxoglutarate dehydrogenase respectively) were measured in heart plus white and fast-oxidative skeletal muscle. No changes were observed in heart muscle. In fast-oxidative skeletal muscle, activities of hexokinase, citrate synthase, and oxoglutarate dehydrogenase were increased by 51, 26, and 33% respectively but there was no effect on 6-phosphofructokinase. These results demonstrate that in red muscle there is no effect of this training programme on the anaerobic capacity but that of the aerobic system is increased by one third. In white skeletal muscle, only the activity of citrate synthase was increased, which indicates that this activity may not provide even qualitative information about changes in capacity of the Krebs cycle.     

Differences in protein mobilization between ventral and dorsal parts of white epaxial muscle from fed, fasted and refed white sturgeon (Acipenser transmontanus)

The effect of starvation-refeeding on protein mobilization in the lateral line, lateral (midway between lateral line and dorsum) and dorsal parts of white sturgeon ( Acipenser transmonlanus ) white epaxial muscle (type IIB) were compared by measuring muscle cross-sectional fibre areas. Effects on red (type I) and pink (type IIA) muscle were also studied. Fish starved throughout the experiment (5 weeks) showed a uniform response, i.e. the cross-sectional fibre area decreased at all sampled locations in the white muscle as well as in red and pink muscle. Fibre size in fish refed for 3 weeks after 2 weeks of fasting (S/F) was reduced mainly in the dorsal region of the white muscle. Starvation for 3 weeks after 2 weeks of initial feeding (F/S) also reduced fibre size mainly in the dorsal regions of the white muscle and in the pink muscle. The results indicate that in sturgeon exposed to short periods of low food availability, protein is utilized preferentially from trie more dorsal regions of the white epaxial muscle and from pink muscle. Protein in white muscle located closer to the lateral line and in red muscle seems to be spared. With the onset of refeeding, protein deposition occurred rapidly in pink muscle, but was delayed significantly in the dorsal part of the white muscle.