Post-mortem glycolysis in ox skeletal muscle. Effect of pre-rigor freezing and thawing on the intermediary metabolism

1. Ox sternomandibularis muscle was ;slow-frozen' by placing it in air at -22 degrees or ;fast-frozen' by immersion in liquid air or acetone-solid carbon dioxide. In all cases muscles were frozen pre-rigor. Changes in length, pH and the concentrations of P(i), creatine phosphate, hexose monophosphate (glucose 1-phosphate+glucose 6-phosphate+fructose 6-phosphate), fructose diphosphate (fructose 1,6-diphosphate+(1/2) triose phosphate), lactate, ATP, ADP, AMP and NAD(+) during freezing and during subsequent thawing were determined. In addition some measurements were made of the changes in alpha-glycerophosphate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate and pyruvate concentrations during slow freezing. 2. Appreciable shortening and marked changes in chemical composition took place during slow freezing but not during fast freezing. 3. During slow freezing the hexose monophosphate concentration fell and fructose 1,6-diphosphate and triose phosphate increased substantially. Increases also took place in 3-phosphoglycerate, 2-phosphoglycerate and phosphoenolpyruvate, but not in pyruvate. 4. On thawing, most of the chemical changes were similar to those in unfrozen muscle post mortem, but took place much more rapidly; loss of NAD(+) was particularly rapid. Fast-frozen muscle metabolized at a faster rate on thawing than did slow-frozen muscle. 5. The overall changes in length during freezing and thawing were about the same in slow-frozen as in fast-frozen muscle.     

Regulation of glycolytic flux in an energetically controlled cell-free system: the effects of adenine nucleotide ratios, inorganic phosphate, pH, and citrate

A soluble extract from rat skeletal muscles has been used with purified mitochondrial ATPase (F₁) to develop steady states with respect to glycolytic flux, the concentrations of glycolytic intermediates and inorganic phosphate, and the concentrations and ratios of adenine nucleotides. Incubations were carried out in media resembling the ionic composition in the cell cytoplasm, in an attempt to evaluate the quantitative contributions of various effectors to the overall control mechanism under simulated in vivo conditions. The primary control reaction of glycolytic flux under the conditions studied could be identified with phosphofructokinase, followed by secondary control of the reaction catalyzed by hexokinase. Glycolytic flux was increased with increasing pH over the range 6.6–7.6, both in the absence and presence of ATPase. Without other added effectors, the glycolyzing extract maintained an ATP/ADP ratio of about 50 in the pH range 7.0–7.6, and phosphofructokinase was incompletely suppressed. Addition of increasing amounts of ATPase markedly stimulated glycolytic flux coincident with lowered steady-state ATP/ADP ratios, and decreased accumulation of hexose monophosphates. Control of flux by the ATP/ADP ratio (and simultaneously altered AMP concentration) was less effective if pH (7.3 to 7.6) or phosphate concentration (2 to 20 mm) was increased. Flux through phosphofructokinase was controlled principally when the ATP/ADP ratios were varied in the range between > 50 and 15. The inhibitory effect of citrate was evaluated. Suppression of glycolytic flux and accumulation of hexose monophosphates were dependent on incubation conditions. If the pH was 7.3 or less, and the phosphate concentration low (2 mm), flux through phosphofructokinase was significantly suppressed even at citrate concentrations less than 50 μm. Simultaneous decrease in the steady-state ATP/ADP ratio and elevation of AMP was ineffective in reversing this inhibition. At higher pH and, more dramatically, when the phosphate concentration was increased, sensitivity to citrate inhibition was markedly diminished. These data, taken together with studies of respiratory control with isolated mitochondria (21., 24.), J. Biol. Chem.250, 2275–2282) strongly suggest that adenine nucleotide control of both glycolysis and respiration is exerted when the ratio of free nucleotides (not protein bound) in the cytosol is in the range of 15 to > 50. The data further suggest that citrate plays an important role in the regulation of glycolysis in muscle when the ATP/ADP ratio is high (and the phosphate concentration is correspondingly low), but that this inhibition is overcome by liberation of inorganic phosphate during muscle contraction.     

The effects of increased heart work on the tricarboxylate cycle and its interactions with glycolysis in the perfused rat heart

1. The work of the perfused rat heart was acutely increased by raising the aortic pressure in the Langendorff preparation from 50 to 120mmHg; within 1 min in perfusions with media containing glucose or glucose+acetate, rates of oxygen consumption and tricarboxylate-cycle turnover increased 2.5-fold, glycolysis rate doubled and oxidation of triglyceride fatty acid was strikingly enhanced. 2. Increased cardiac work had no significant effects on the heart concentrations of creatine phosphate, ATP, ADP or 5′-AMP. The only significant changes in tricarboxylate-cycle intermediates were a decrease in malate in perfusions with glucose and decreases in acetyl-CoA and citrate and an increase in aspartate in perfusions with glucose+acetate. 3. Measurements of intracellular concentrations of hexose phosphates, glucose and glycogen indicated that work accelerated glycolysis by activation of phosphofructokinase and subsequently hexokinase; the activation could not be accounted for by changes in the known effectors of phosphofructokinase. 4. Acetate at either perfusion pressure increased heart concentrations of acetyl-CoA, citrate, glutamate and malate and decreased that of aspartate; acetate increased tricarboxylate-cycle turnover by 50–60% and inhibited glycolysis and pyruvate oxidation. 5. In view of the markedly different effects of acetate and of cardiac work on the concentrations of cycle intermediates the changes that accompany acetate utilization may be specifically concerned with the regulatory functions of the cycle in control of glycolysis and pyruvate oxidation and not with the associated increase in cycle turnover. It is suggested that the concentrations of key metabolites controlling the rate of cycle turnover may fluctuate with each heart beat and that this may explain why no significant changes (for example, in adenine nucleotide concentrations) have been detected with increased work in the present study.     

Some effects of glucose concentration and anoxia on glycolysis and metabolite concentrations in the perfused liver of fetal guinea pig.

Effects of glucose concentration and anoxia upon the metabolite concentrations and rates of glycolysis and respiration have been investigated in the perfused liver of the fetal guinea pig. In most cases the metabolite concentrations in the perfused liver were similar to those observed in vivo. Between 50 days and term there was a fall in the respiratory rate and in the concentration of ATP and fructose 1,6-diphosphate and an increase in the concentration of glutamate, glycogen and glucose. Reducing the medium glucose concentration from 10 mM to 1 mM or 0.1 mM depressed lactate production and the concentration of most of the phosphorylated intermediates (except 6-phosphogluconate) in the liver of the 50-day fetus. This indicates a fall in glycolytic rate which is not in accord with the known kinetic properties of hexokinase in the fetal liver. Anoxia increased lactate production by, and the concentrations of, the hexose phosphates ADP and AMP in the 50-day to term fetal liver, while the concentration of ribulose 5-phosphate, ATP and some triose phosphates fell. These results are consistent with an activation of glycolysis, particularly at phosphofructokinase and of a reduction in pentose phosphate pathway activity, particularly at 6-phosphogluconate dehydrogenase. The calculated cytosolic NAD+/NADH ratio for the perfused liver was similar to that measured in vivo and evidence is presented to suggest that the dihydroxyacetone phosphate/glycerol 3-phosphate ratio gives a better indication of cytosolic redox than the lactate/pyruvate ratio. The present observations indicate that phosphofructokinase hexokinase and possibly pyruvate kinase control the glycolytic rate and that glyceraldehyde-3-phosphate dehydrogenase is at equilibrium in the perfused liver of the fetal guinea pig.     

Some effects of glucose concentration and anoxia on glycolysis and metabolite concentrations in the perfused liver of fetal guinea pig

Effects of glucose concentration and anoxia upon the metabolite concentrations and rates of glycolysis and respiration have been investigated in the perfused liver of the fetal guinea pig. In most cases the metabolite concentrations in the perfused liver were similar to those observed in vivo. Between 50 days and term there was a fall in the respiratory rate and in the concentration of ATP and fructose 1,6-diphosphate and an increase in the concentration of glutamate, glycogen and glucose. Reducing the medium glucose concentration from 10 mM to 1 mM or 0.1 mM depressed lactate production and the concentration of most of the phosphorylated intermediates (except 6-phosphogluconate) in the liver of the 50-day fetus. This indicates a fall in glycolytic rate which is not in accord with the known kinetic properties of hexokinase in the fetal liver. Anoxia increased lactate production by, and the concentrations of, the hexose phosphates ADP and AMP in the 50-day to term fetal liver, while the concentration of ribulose 5-phosphate, ATP and some triose phosphates fell. These results are consistent with an activation of glycolysis, particularly at phosphofructokinase and of a reduction in pentose phosphate pathway activity, particularly at 6-phosphogluconate dehydrogenase.The calculated cytosolic NAD⁺/NADH ratio for the perfused liver was similar to that measured in vivo and evidence is presented to suggest that the dihydroxyacetone phosphate/glycerol 3-phosphate ratio gives a better indication of cytosolic redox than the lactate/pyruvate ratio. The present observations indicate that phosphofructokinase and hexokinase and possibly pyruvate kinase control the glycolytic rate and that glyceraldehyde-3-phosphate dehydrogenase is at equilibrium in the perfused liver of the fetal guinea pig.     

Citrate and the regulation of adipose-tissue phosphofructokinase

1. Methods are described for the extraction, partial purification and assay of phosphofructokinase in rat epididymal adipose tissue. 2. The enzyme was inhibited by ATP at concentrations above 20mum and by citrate; the enzyme was activated by ADP, AMP, 3',5'-(cyclic)-AMP, phosphate and sulphate. 3. The enzyme lost activity on incubation at 25 degrees or during chromatography on DEAE-cellulose unless fluoride at a concentration of 4mm was present. 4. The significance of these results in relation to the role of glycolysis and citrate in lipogenesis is discussed.     

Interaction of immobilized phosphofructokinase with soluble muscle proteins

Selected glycolytic enzymes (including phosphoglucose isomerase, aldolase, glyceraldehyde phosphate dehydrogenase, enolase, pyruvate kinase and lactate dehydrogenase), as well as glycogen phosphorylase, creatine kinase, and adenylate kinase, bound to phosphofructokinase immobilized on an agarose gel. The affinity of phosphofructokinase to these various proteins differed, with phosphorylase exhibiting the strongest binding. Binding was reversed either by: (1) elution with high-ionic-strength buffer (0.4 M KCl); (2) the addition of a 5-10 mM concentration of ATP; or (3) high concentrations of fructose 6-phosphate (5 mM).     

31P-NMR studies of phosphate metabolites in intact red and white swimming muscles of cod (Gadus morhua L.)

31P-NMR was used to characterise intracellular phosphate pools and their post mortem changes at 7 degrees C in intact red and white cod muscles under anaerobic conditions. A total phosphate content of 55 and 60 mM was observed in red and white muscle, respectively. The concentration of P-creatine was 14 mM in the white and 9 mM in the red muscle, while that of inorganic phosphate, Pi (30 mM), ATP (9 mM), and sugar phosphate (5 mM) were similar in both muscles. During the first 90 min after death, the decrease in P-creatine showed a first order breakdown with a concomitant stoichiometric increase in Pi content, whereas the ATP and sugar phosphate remained the same. The intracellular pH decreased from 7.4 to 7.3 in this period. The steady-state rate constant of myosin ATPase was 0.0054 and 0.0022/min for red and white muscles, respectively. Individuals kept under diminished oxygen tension prior to being killed, showed a reduced P-creatine level in both muscles.     

Control of glycolysis by orthophosphate and adenosine triphosphate in soluble extracts of germinating pea seeds

Summary Control of aerobic glycolysis by adenosine triphosphate and orthophosphate has been studied in cell-free extracts of germinating pea seeds. Orthophosphate accelerates glycolysis under all conditions studied. At high concentrations of magnesium ion ATP accelerates glycolysis, whereas at lower magnesium concentrations ATP severely inhibits glycolysis. The inhibitory effect of ATP is markedly relieved by orthophosphate. Metabolite analyses suggest an important regulatory role of phosphofructokinase and show that low ratios of F-6-P: FDP accompany the appearance of a high rate of glycolysis, and vice versa. Thus, ATP raises the F-6-P: FDP ratio at low magnesium levels, while P_i lowers this ratio. At high Mg²⁺ (where ATP accelerates glycolysis), ATP causes a low F-6-P: FDP ratio to appear. At low Mg²⁺ concentration, orthophosphate accelerates glycolysis by activation of phosphofructokinase; at high magnesium concentration, the chief effect of orthophosphate is its long-known role in facilitating the oxidation of triose phosphate.     

Different rates of glycolysis affect glycolytic activities and protein properties in turkey breast muscle

Protein alterations of turkey breast muscles (Pectoralis major) were investigated at 20 min and 24 h post mortem. Specific activities, quantities and kinetic parameters of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and aldolase A were also determined at 20 min post mortem. Based on the pH values at 20 min post mortem, two groups of samples were classified as rapid glycolysis group (RG; pH20 min = 5.80 ± 0.07, n = 20) and normal glycolysis group (NG; pH20 min = 6.21 ± 0.01, n = 20). RG had lower specific activities of GAPDH and aldolase A than NG while Vm and Km values of both enzymes were not different between groups. RG showed lower high ionic strength (HIS) and pellet protein extractabilities at 20 min post mortem. It also had lower low ionic strength (LIS) and HIS protein extractabilities at 24 h post mortem. Besides pellet protein, muscular protein extractabilities at 24 h post mortem were higher than at 20 min post mortem. From SDS-PAGE of samples at 24 h post mortem, RG exhibited lower band intensities at 45 and 200 kDa, which were further identified as actin and myosin heavy chain (MHC), respectively. Western blots revealed that relative amounts of actin and MHC at 20 min post mortem were not different between groups. However, RG muscles had less relative amount of actin at 24 h post mortem. It also indicated that amounts of actin and MHC increased with regard to post mortem time.     

31P NMR study of phosphorus metabolites in fast and slow muscles

1. 31P NMR was used to characterize phosphate pools in perchloric acid extracts of muscles with various composition of muscle fibre types. 2. The white m. pectoralis major (MPM) of chickens 15 min post mortem is characterized by 1.6-times higher relative content of phosphocreatine (PCr) in comparison with mixed leg muscle (LM) of this species. The glycerophosphorylcholine (GPC) does not occur in MPM at NMR detectable level in contrast to the leg muscles. Relative amounts of other phosphates are similar in both muscles. 3. The intermediate MPM of pigeons as well as mixed LM of this species contain 15 min post mortem a very small amount of PCr and ATP but a large amount of inorganic phosphate. Relative content of GPC is higher in leg muscles than in intermediate MPM. 4. Muscles with higher occurrence of white fibres contain relatively more PCr than muscles with lower occurrence of white fibres. 5. The occurrence of GPC seems to be connected with metabolism of red muscle fibres.     

The effects of ammonium, inorganic phosphate and potassium ions on the activity of phosphofructokinases from muscle and nervous tissues of vertebrates and invertebrates.

1. The effect of NH4+, Pi and K+ on phosphofructokinase from muscle and nervous tissues of a large number of animals was investigated. The activation of the enzyme from lobster abdominal muscle by NH4+ was increased synergistically by the presence of Pi or SO4(2-). In the absence of K+, NH4+ plus Pi markedly activated phosphofructokinase from all tissues studied. In the presence of 100 mM-K+, NH4+ plus Pi activated phosphofructokinase from nervous tissue and muscle of invertebrates and the enzyme from brain of vertebrates, but there was no effect of NH4+ plus Pi on the enzyme from the muscles of vertebrates. Nonetheless, NH4+ plus Pi increased the activity of vertebrate muscle phosphofructokinase in the presence of 50 mM-K+ at inhibitory concentrations of ATP, i.e. these ions de-inhibited the enzyme. In the absence of NH4+ plus Pi, K+ activated phosphofructokinase from vertebrate tissues at non-inhibitory ATP concentrations, but the effect was less marked with the enzyme from invertebrate tissues. Indeed, high concentrations of K+ (greater than 50 mM) caused inhibition of invertebrate tissue phosphofructokinase. Of the other alkali-metal ions tested, only Rb+ activated phosphofructokinase from lobster abdominal muscle and rat heart muscle. 2. The properties of lobster abdominal-muscle phosphofructokinase were studied in detail. This muscle was chosen as representative of invertebrate muscle because large quantities of tissue could be obtained from one animal and the enzyme was considerably more stable in tissue extracts than in extracts of insect flight muscle. In general, the properties of the enzyme from this tissue were similar to those of the enzyme from many other tissues: ATP concentrations above an optimum value inhibited the enzyme and this inhibition was decreased by raising the fructose 6-phosphate or the AMP concentration. In particular, NH4+ plus Pi activated the enzyme at noninhibitory concentrations of ATP and they also relieved ATP inhibition (see above). 3. It is suggested that increases in the concentration of NH4+ and Pi, under conditions of increased ATP utilization in certain muscles and/or nervous tissue, may play a part in the stimulation of glycolysis through the effects on phosphofructokinase (the effect may be a direct activation and/or a relief of ATP inhibition). Changes in the concentration of NH4+ and Pi are consistent with this theory in nervous tissue and the anaerobic type of muscles. The role of AMP deaminase in production of NH4+ from AMP in these tissues is discussed in relation to the control of glycolysis.     

Phosphorus-31 nuclear magnetic resonance study of post mortem catabolism and intracellular pH in intact excised rabbit muscle

Phosphorus-31 nuclear magnetic resonance has been used to study the post mortem catabolism of high-energy phosphate compounds and the associated intracellular pH variation in pure fast- and slow-twitch rabbit muscles and in rabbit muscle with mixed fiber types. Comparative results from pure fiber types are reported for the first time. Large amounts of glycerophosphorylcholine (14.1 mumol/g fresh tissue) are found in the internal conoidal bundle (ICB), a pure oxidative slow twitch muscle, whereas the m. psoas major (PM), a pure glycolytic fast twitch muscle and the m. gastrocnemius caput medialis (GCM), with mixed fiber types, are devoid of the same metabolite. The total content of phosphorylated metabolites is constant among the three muscle types. The time-dependent post mortem changes in phosphorylated metabolites display the expected rapid drop in phosphocreatine and a simultaneous increase in intracellular inorganic phosphate. However, the ATP level remains constant during more than 2 h. Rate constants for metabolite breakdown and apparent ATPase activity have been determined. The comparative kinetics of intracellular acidosis at 25 degrees C yield rates of 3.3 X 10(-3) pH unit/min for PM, 2.7 X 10(-3) pH unit/min for GCM and 3.0 X 10(-3) pH unit/min for ICB. Initial intracellular pH values are 7.07, 7.20 and 7.02, respectively. Upon aging, the heterogeneity of the Pi signal reflects the existence of cellular compartments with different internal pH. The results suggest that the more intense low-pH Pi signal arises from the sarcoplasmic reticulum while the less intense resonance would reflect the sarcoplasmic higher pH. The temperature effect on post mortem catabolism in the 15-25 degrees C range has been documented. As expected, phosphocreatine and ATP breakdown increase with temperature but at a higher rate for slow-twitch ICB than for fast-twitch PM.     

Studies on the mechanism of the antifungal action of benzoate.

A method is described for the determination of the pH of intracellular water based on the distribution of [14C]benzoate (0.01 mM) between intra- and extra-cellular water. Benzoate at higher concentrations (2-10mM) enters the yeast cell in the undissociated form, and its neutralization within the cell can cause a shift of the pH of the intracellular water by more than 1 pH unit. Benzoate causes an accumulation of the two hexose monophosphates of yeast glucose fermentation and a decrease in intermediates beyond phosphofructokinase, suggesting inhibition at this stage. Benzoate also causes a concomitant fall in [ATP]. Phosphofructokinase is inhibited to a greater extent than hexokinase at acid pH. There is a relationship between intracellular pH, phosphofructokinase inhibition and CO2 production, suggesting that the antifungal action of benzoate is caused by an accumulation of benzoate at low external pH, which lowers the intracellular pH into the range where phosphofructokinase is sensitive. The subsequent inhibition of glycolysis causes a fall in [ATP] and thus restricts growth.     

Temperature regulation of glucose metabolism in red blood cells of the freeze-tolerant wood frog.

The low-temperature metabolism of erythrocytes from the freeze-tolerant frog Rana sylvatica was investigated by (13)C and (31)P NMR spectroscopy. Erythrocytes readily took up high concentrations of the natural cryoprotectant, glucose, at both high (12 and 17 degrees C) and low (4 degrees C) temperatures but glucose was apparently not metabolized at 4 degrees C. Strong inhibition of glucose catabolism at low temperature would facilitate the maintenance of the very high concentrations of glucose (approximately 200 mM) that are accumulated to provide cryoprotection during freezing in wood frogs. Analysis of (13)C labeling of glycolytic intermediates at 4 degrees C showed mixing of label primarily in hexose (fructose) and hexose phosphate (glucose 6-phosphate, fructose 6-phosphate) pools but little label incorporation into triose phosphate intermediates. These data are consistent with a profound low-temperature-induced inhibition of phosphofructokinase (PFK). Investigations into potential PFK control mechanisms were undertaken. (31)P NMR analysis showed that the intracellular pH of erythrocytes increased from 7.0 to 7.3 as temperature decreased from 17 to 4 degrees C in a manner consistent with alphastat regulation. This change is exactly opposite to that expected if overall PFK activity was regulated by changes in cellular pH since PFK is less active at lower pH values in vitro. Other factors must, therefore, operate to regulate PFK at lower temperatures.     

The control of glycogen metabolism in yeast. 1. Interconversion in vivo of glycogen synthase and glycogen phosphorylase induced by glucose, a nitrogen source or uncouplers

The addition of glucose to a suspension of yeast initiated glycogen synthesis and ethanol formation. Other effects of the glucose addition were a transient rise in the concentration of cyclic AMP and a more prolonged increase in the concentration of hexose 6-monophosphate and of fructose 2,6-bisphosphate. The activity of glycogen synthase increased about 4-fold and that of glycogen phosphorylase decreased 3-5-fold. These changes could be reversed by the removal of glucose from the medium and induced again by a new addition of the sugar. These effects of glucose were also obtained with glucose derivatives known to form the corresponding 6-phosphoester. Similar changes in glycogen synthase and glycogen phosphorylase activity were induced by glucose in a thermosensitive mutant deficient in adenylate cyclase (cdc35) when incubated at the permissive temperature of 26 degrees C, but were much more pronounced at the nonpermissive temperature of 35 degrees C. Under the latter condition, glycogen synthase was nearly fully activated and glycogen phosphorylase fully inactivated. Such large effects of glucose were, however, not seen in another adenylate-cyclase-deficient mutant (cyr1), able to incorporate exogenous cyclic AMP. When a nitrogen source or uncouplers were added to the incubation medium after glucose, they had effects on glycogen metabolism and on the activity of glycogen synthase and glycogen phosphorylase which were directly opposite to those of glucose. By contrast, like glucose, these agents also caused, under most experimental conditions, a detectable rise in cyclic AMP concentration and a series of cyclic-AMP-dependent effects such as an activation of phosphofructokinase 2 and of trehalase and an increase in the concentration of fructose 2,6-bisphosphate and in the rate of glycolysis. Under all experimental conditions, the rate of glycolysis was proportional to the concentration of fructose 2,6-bisphosphate. Uncouplers, but not a nitrogen source, also induced an activation of glycogen phosphorylase and an inactivation of glycogen synthase when added to the cdc35 mutant incubated at the restrictive temperature of 35 degrees C without affecting cyclic AMP concentration.     

The reliability of histochemical fibre typing of human necropsy muscles

The reliability of muscle fibre typing of post mortem specimens was investigated with special reference to the influence of time and temperature. In specimens stored at +4 degrees C, muscle fibre typing could be reliably performed up to at least ten and fifteen days post mortem for the masseter and biceps brachii muscles respectively. The corresponding figures for storage at room temperature were three and six days. The difference in the preservation of enzyme activity between masticatory and limb muscles might be related to the demonstrated difference in the fibre type composition and thus the enzyme content and energy sources.     

The contents of adenine nucleotides, phosphagens and some glycolytic intermediates in resting muscles from vertebrates and invertebrates.

The lowest contents of ATP and the lowest ATP/AMP concentration ratios are observed in the molluscan muscles that have very low rates of energy expenditure during contraction. The highest contents of ATP are observed in the extremely aerobic insect flight muscle and the extremely anaerobic pectoral muscle of the pheasant and domestic fowl. In general, the lowest ATP/AMP concentration ratios are observed for muscle in which the variation in the rate of energy utilization is small (e.g. some molluscan muscles, heart muscle); the highest ratios are observed in muscles in which this variation is large (lobster abdominal muscle, pheasant pectoral muscle, some insect flight muscles). This finding is consistent with the proposed role of AMP and the adenylate kinase reaction in the regulation of glycolysis. However, in the flight muscle of the honey-bee the ATP/AMP ratio is very low, so that glycolysis may be regulated by factors other than the variation in AMP concentration. The variation in the contents of arginine phosphate in muscle from the invertebrates is much larger than the variation in creatine phosphate in muscle from the vertebrates. The contents of hexose monophosphates and pyruvate are, in general, higher in the muscles of vertebrates than in those of the invertebrates. The contents of phosphoenolpyruvate are similar in all the muscles investigated, except for the honey-bee in which it is about 4-10-fold higher. The mass-action ratios for the reactions catalysed by phosphoglucoisomerase and adenylate kinase are very similar to the equilibrium constants for these reactions. Further, the variation in the mass-action ratios between muscles is small. It is concluded that these enzymes catalyse reactions close to equilibrium. However, the mass-action ratios for the reactions catalysed by phosphofructokinase and pyruvate kinase are much smaller than the equilibrium constants. The variation in the ratios between different muscles is large. It is concluded that these enzymes catalyse nonequilibrium reactions. Since the variation in the mass-action ratios for the reactions catalysed by the phosphagen kinases (i.e. creatine and arginine phosphokinases) is small, it is suggested that these reactions are close to equilibrium.     

The effect of inosine, inorganic phosphates and pyruvate on erythrocyte glycolysis metabolites under conditions of preservation]

Human erythrocytes were stored as resuspensions in solutions containing citrate (Z), inosine + citrate (I), inosine + phosphate (IP), and inosine + phosphate + pyruvate (IPP). The storage was made at + 4 degrees C for 6 weeks; the initial pH-value amounted to 7.4 at + 4 degrees C. The cellular concentrations of 2.3 DPG, ATP, G6P, FDP and DOAP + GAP were determined. The following results were obtained: 1. During the storage in stored Z-blood the 2.3 DPG concentration will fall below 10% of its initial value; it will remain nearly unchanged in stored I-blood and will increase to 170% in stored IP-blood, to 270% of its initial value in stored IPP-blood. 2. The ATP concentration of cells will fall to about 50% of its initial value at the beginning of the storage of all stored blood. After that it will only increase to about 80% of its initial value in stored IP- and IPP-blood. 3. During the storage the G6P concentration will increase to the highest degree in stored IPP-blood and if high pyruvate concentrations are not present, it will have a reciprocal behaviour towards the FDP and triosephosphate level. The results were discussed in view of the regulation of glycolysis under storage conditions.     

Metabolic characteristics of muscles in the spiny lobster, Jasus edwardsii, and responses to emersion during simulated live transport

The metabolic characteristics of five muscle groups in the spiny lobster Jasus edwardsii were examined in order to compare their anaerobic and oxidative capacities. Enzyme activities of phosphorylase, phosphofructokinase, pyruvate kinase, and lactate dehydrogenase were highest in abdominal muscles supporting anaerobic burst activity. Hexokinase, citrate synthase, and HOAD activities in the leg and antennal muscles indicated higher aerobic potential. Arginine kinase activities were high in all muscle groups indicating that muscle phosphagens are an important energy reserve. Arginine phosphate concentrations in 4th periopod and abdominal flexor muscle from lobsters sampled in the field were higher than any values from captive animals, and approximately five times those for ATP. Muscle lactates were high in captive animals. Responses to emersion during simulated live transport appear to exploit the capacity for functional anaerobiosis and further differentiated the muscle groups. Abdominal muscles were especially sensitive and after 24 h showed significant increases in lactate, glucose, ADP, and AMP. ATP levels appeared to be maintained by muscle phosphagens and raised doubts about the efficacy of the adenylate energy charge in evaluating the emersion response. Haemolymph glucose, lactic acid, and ammonia peaked after 24 h emersion and were largely restored following re-immersion. We propose that arginine phosphate concentrations in the 4th periopod are an appropriate index of metabolic stress, and could lead to improved commercial handling protocols.