Control of glycolysis in the posterior adductor muscle of the sea musselMytilus edulis

Summary Concentrations of glycolytic intermediates, lactate, adenine nucleotides, inorganic phosphate, phosphoarginine and citrate have been estimated after various periods of valve closure (Table 1 and Fig. 1). Mass action ratios of enzyme steps involved in the metabolism of these components are compared with their equilibrium constants. This reveals glycogen phosphorylase, phosphofructokinase, hexosediphosphatase and pyruvate kinase catalyze non-equilibrium reactions. The first three enzymes possess relatively low activities (Table 2).From the changes in concentrations of the glycolytic intermediates it is concluded that phosphofructokinase controls the carbon flow during the first hours after valve closure, whereas later on the rate of conversion of phosphoenolpyruvate is determining this flow. In skeletal muscle phosphofructokinase controls the carbon flow during the whole period of exercise.The concentrations of ADP, AMP and inorganic phosphate increase, whereas the concentrations of ATP, phosphoarginine and citrate decrease during valve closure (Table 1 and Fig. 2). In contrast to skeletal muscle, these changes do not result in a strong increase in the glycolytic flux.There is a much greater potential for ATP hydrolysis by the myofibrillar ATPase system than is actually realized by the adductor muscle during valve closure.     

Flux regulation in glycogen-induced oscillatory glycolysis in cell-free extracts of Saccharomyces carlsbergensis

To localise the controlling point of the glycolytic system, the temporal changes of concentrations of glycolytic intermediates have been analysed after addition of glycogen to a substrate-depleted yeast extract. Three sequential metabolic states are clearly observable: a transition state at which there is continuous accumulation of the intermediates before the glyceraldehydephosphate dehydrogenase (GAPDH, EC 1.2.1.12) step; a stationary state with all glycolytic intermediates having concentrations oscillating at nearly stationary mean values; and a depletion state at which the intermediates before the GAPDH step are being depleted due to the exhaustion of glycogen. In all these states, the mean ethanol production rate and the concentration of ATP and the intermediates beyond the GAPDH-step are maintained fairly constant, while the glycogen consumption rate and intermediate concentrations of the upper part of the glycolytic system change considerably: the glycogen consumption rate varies 4-fold and fructose-bis-phosphate concentration more than 10-fold. Doubling of the initial glycogen concentration and the addition of a great excess of fructose-bis-phosphate do not affect the ethanol production rate and the mean glycerate-3-phosphate (3-PGA) and pyruvate levels. By contrast, ethanol production was accelerated by an increase of the net ATP consumption rate resulting from either the addition of apyrase or by substitution of trehalose for glycogen. Neither the mean absolute ATP level nor the adenylate energy charge were measurably affected, however all this data can be interpreted in terms of a very strong stoichiometric regulation and stabilization of the lower part of the glycolytic system.     

24-h organization of glycolysis and control by photoperiodism

A study of the temporal organization of glycolysis at diverse levels of activity of the pathway showed that consideration of the phases of the 24h oscillations in glycolytic pools affords a means of detecting modifications in regulatory mechanisms according to the level of carbon flow along the pathway. The work utilized Kalanchoe blossfeldiana, a plant with crassulacean acid metabolism (CAM) in which glycolytic activity is under the control of photoperiodism: after transfer from long days to short days carbon flow through the pathway increases drastically. Analysis of the glycolytic pools performed during the day/night cycle showed that: a. 24 h-period variations exist in the content of the glycolytic intermediates; b. time of the acrophase of these rhythms changes as a function of the photoperiodic treatment: in long days the pools of the intermediates preceding the phosphofructokinase (PFK) step oscillate in phase and the same holds for the intermediates after the PFK step but these two sequences of the pathway oscillate out of phase (phase-jump of about 10h); transfer to short days besides producing (after a lag) changes in the mean level and amplitude of the oscillations, modifies their phase: this temporal reorganization of glycolysis results in splitting the pathway into 3 sequences of synchronously-oscillating pools, phase-jumps between successive sequences occurring at the PFK (4h) and at the 3-phosphoglyceraldehyde dehydrogenase (12h) steps.     

Motion of both mitral valve leaflets: a cineroentgenographic study in intact dogs.

Motion and position of both mitral leaflets were studied in five normal dogs 1-11 wk after radiopaque markers were sutured on the valve cusps and on the mitral annulus. Cinefluorograms and cineangiograms (100-120 frames/s) of left atrium and left ventricle were used to study cusp motion and intraventricular flow patterns, and to detect mitral regurgitation during sinus rhythm (42-184 beats/min) and during isolated atrial or ventricular contractions. Time-motion of both leaflets was similar throughout diastole with the exception of delayed posterior cusp opening. Peak opening and closing speeds, opening and closing times, and time of complete closure, measured from the Q wave of the ECG, were not significantly affected by the variations in heart rate. Diastolic leaflet closure began immediately after opening, while the ventricular cavity was small, and was caused by flow eddies behind the cusps. Isolated ventricular contractions closed the valve leaflets completely and symmetric valve closure was ensured by the different rates of leaflet edge approximation. In contrast, atrial closure was slow, partial, and of very short duration.     

Effects of acute asphyxia on brain energy metabolism in fetal guinea pigs near term.

In a previous study we suggested that--unlike other forms of asphyxia--acute asphyxia caused by arrest of uterine blood flow is accompanied by a fall in oxygen delivery to the fetal brain (Jensen et al., 1987). This may change cerebral energy metabolism by causing an increase in the glycolytic rate. To test this hypothesis we studied the time course of the changes in the levels of high-energy phosphates and glycolytic intermediates in the cerebral cortex of unanaesthetized fetal guinea pigs near term before and after 2 and 4 min of acute asphyxia. During asphyxia there was a progressive fall of adenosine triphosphate, creatine-phosphate, glucose and fructose-1,6-diphosphate concentrations, whereas adenosine diphosphate, adenosine monophosphate and lactate concentrations increased. Pyruvate concentrations did not change. We conclude that fetal cerebral energy metabolism becomes increasingly anaerobic during acute asphyxia caused by arrest of uterine blood flow, because oxygen delivery to the fetal brain falls.     

Phytohaemagglutinin stimulation of rat thymus lymphocytes glycolysis.

Glucose disappearance and lactate production by the rat thymocytes are stimulated significantly 45 min after addition of phytohaemagglutinin or concanavalin A and the stimulated rate is sustained for at least 8 h. Changes in the steady-state concentration of glycolytic intermediates that occur at non-equilibrium steps during the increased rate of glycolytic flux indicate that the glucose carrier, hexokinase and phosphofructokinase are potentially regulatory steps that undergo nearly simultaneous or tightly sequential activation following interaction of the cells with the mitogen.     

Phytohaemagglutinin stimulation of rat thymus lymphocyte glycolysis

Glucose disappearance and lactate production by rat thymocytes are stimulated significantly 45 min after addition of phytohaemagglutinin or concanavalin A and the stimulated rate is sustained for at least 8 h.Changes in the steady-state concentration of glycolytic intermediates that occur at non-equilibrium steps during the increased rate of glycolytic flux indicate that the glucose carrier, hexokinase and phosphofructokinase are potentially regulatory steps that undergo nearly simultaneous or tightly sequential activation following interaction of the cells with the mitogen.     

Comparative in vitro study of bileaflet and tilting disk valve behavior in the pulmonary position

A study of mechanical heart valve behavior in the pulmonary position as a function of pulmonary vascular resistance is reported for the St. Jude Medical bileaflet (SJMB) valve and the MedicalCV Omnicarbon (OTD) tilting disk valve. Tests were conducted in a pulmonic mock circulatory system and impedance was varied in terms of system pulmonary vascular resistance (PVR). An impedance spectrum was found using instantaneous pulmonary artery pressure and flow rate curves. Both valves fully opened and closed at and above a nominal PVR of 3.0 mmHg/L/min. The SJMB valve was prone to leaflet bounce at closure, but otherwise completely closed, at settings above and below this nominal setting. At PVR values at and below 2.0 mmHg/L/min, the SJMB valve exhibited two types of leaflet aberrant behavior: single leaflet only closure while the other leaflet fluttered, and incomplete closure where both leaflets flutter but neither remain fully closed. The OTD valve fully opened and closed to a PVR value of 1.6 mmHg/L/min. At lower values, the valve did not close. Valves designed for the left heart can show aberrant behavior under normal conditions as pulmonary valves.     

Dynamic 13C-tracer study of storage carbohydrate pools in aerobic glucose-limited Saccharomyces cerevisiae confirms a rapid steady-state turnover and fast mobilization during a modest stepup in the glucose uptake rate

In this research, two dynamic ¹³C-labeling experiments confirmed turnover and rapid mobilization of stored glycogen and trehalose in an aerobic glucose-limited chemostat ( D =0.05 h⁻¹) culture of Saccharomyces cerevisiae . In one experiment, the continuous feed to an aerobic glucose-limited chemostat culture of S. cerevisiae was instantaneously switched from naturally labeled to fully ¹³C labeled while maintaining the same feed rate before and after the switch. The dynamic replacements of naturally labeled intracellular glycolytic intermediates and CO₂ (in the off-gas) with their ¹³C-labeled equivalents were measured. The data of this experiment suggest that the continuous turnover of glycogen and trehalose is substantial ( c . 1/3 of the glycolytic flux). The second experiment combined the medium switch with a shiftup in the glucose feeding rate (dilution rate shiftup from 0.05 to 0.10 h⁻¹). This experiment triggered a strong but transient mobilization of storage carbon, that was channelled into glycolysis, causing a significant disruption in the dynamic labeling profile of glycolytic intermediates. The off-gas measurements in the shiftup experiment confirmed a considerable transient influx of ¹²C-carbon into glycolysis after the combined medium switch and dilution rate shiftup. This study shows that for accurate in vivo kinetic interpretation of rapid pulse experiments, glycogen and trehalose metabolism must be taken into account.     

Studies on cerebral energy metabolism during the course of galactose neurotoxicity in chicks

—At various times during a 2-day study, the levels of adenine nucleotides and selected glycolytic intermediates were determined in brains of chicks fed a diet containing d -galactose (40%, w/w). The levels of ATP and glucose 6-phosphate had decreased by 9 h after initiation of the diet, whereas those of fructose 1,6-diphosphate, 3-phosphoglycerate, l -α-glycerophosphate, and lactate were not reduced until after 18 h had elasped. Although glucose 1-phosphate was not appreciably affected, glucose and glycogen were depleted during the latter stages of the toxicity. The cerebral levels of 3′,5′-cyclic AMP and citrate did not differ significantly between the two dietary groups at 48 h. The changes in the levels of cerebral glycolytic intermediates and high-energy phosphates during ischemia indicated that the glycolytic rate was diminished in the chicks fed galactose and that high-energy phosphate compounds were depleted sooner than in controls. After intraperitoneal injection of [¹⁴C]glucose, the specific radioactivity and levels of glucose in the plasma from chicks fed either diet were similar, whereas they were significantly reduced in the brains from galactosefed animals. We suggest that galactose interferes with the uptake of glucose into the brain and that this mechanism may be an important factor in d -galactose-induced neurotoxicity in the chick.     

The regulation of glycolysis in perfused locust flight muscle

Concentrations of glycolytic intermediates, amino acids and possible regulator substances were measured in extracts from locust thoracic muscles perfused under different conditions. The conversion of [(14)C]glucose into intermediates and CO(2) by muscle preparations was also followed. When muscles perfused with glucose were made anaerobic changes in metabolite concentrations occurred that could be accounted for by an activation of phosphofructokinase and pyruvate kinase. When butyrate and glucose were present in the perfusion medium the rate of glycolytic flux was lower than with glucose alone, and the aldolase reaction appeared to be inhibited. When butyrate alone was supplied to the muscle the concentrations of most glycolytic intermediates were similar to those found when glucose was supplied. Iodoacetate caused changes in concentrations of intermediates that appeared to result from inhibition of glyceraldehyde 3-phosphate dehydrogenase. Fluoroacetate-poisoned muscles showed a high citrate concentration, but no obvious site of inhibition by citrate was apparent in the glycolytic pathway. Mechanisms for control of glycolysis in locust flight muscle are discussed and related to the known properties of isolated enzymes. It is proposed that trehalase, hexokinase, phosphofructokinase, aldolase, and pyruvate kinase may be control enzymes in this tissue.     

Inorganic Phosphate (Pi) Enhancement of Dark Respiration in the Pi-Limited Green Alga Selenastrum minutum (Interactions between H+/Pi Cotransport,the Plasmalemma H+-ATPase,and Dark Respiratory Carbon Flow)

Inorganic phosphate (Pi) enrichment of the Pi-limited green alga Selenastrum minutum in the dark caused a 2.5-fold increase in the rate of O2 consumption. Alkalization of the media during Pi assimilation was consistent with a H+/Pi cotransport mechanism with a stoichiometry of at least 2 H+ cotransported per Pi. Dark O2 consumption remained enhanced beyond the period of Pi assimilation and did not recover until the medium was reacidified. This result, coupled with an immediate decrease in adenylate energy charge following Pi enrichment, suggested that respiration is regulated by the ATP requirements of a plasmalemma H+-ATPase that is activated to maintain intracellular pH and provide proton motive force to power Pi uptake. Concentrations of tricarboxylic acid cycle intermediates decreased following Pi enrichment and respiratory CO2 efflux increased, indicating that the tricarboxylic acid cycle was activated to supply reductant to the mitochondrial electron transport chain. These results are consistent with direct inhibition of electron transport by ADP limitation. Enhanced rates of starch breakdown and increases in glycolytic metabolites indicated that respiratory carbon flow was activated to supply reductant to the electron transport chain and to rapidly assimilate Pi into metabolic intermediates. The mechanism that initiates glycolytic carbon flow could not be clearly identified by product:substrate ratios due to the complex nature of Pi assimilation. High levels of triose-P and low levels of phosphoenolpyruvate were the primary regulators of pyruvate kinase and phosphofructokinase, respectively.     

Regulatory steps of glycolysis during environmental anoxia and muscular work in the cockle,Cardium tuberculatum: control of low and high glycolytic flux

Summary Concentrations of glycolytic intermediates, end products of anaerobic metabolism and the adenylates have been determined in the foot muscle and in the whole soft body tissue of the cockle,Cardium tuberculatum, after anoxic incubation and after the performance of vigorous escape movements. Comparison of the mass action ratios (MAR) with the equilibrium constants (Keq) showed that the reactions catalyzed by glycogen phosphorylase, hexokinase, phosphofructokinase (PFK) and pyruvate kinase (PK) were displaced from equilibrium under all physiological situations investigated.Changes in the levels of the glycolytic intermediates showed that activation of phosphofructokinase is largely responsible for the 100-fold increase of glycolytic flux in the foot muscle during exercise.Analysis of the whole soft body tissue showed that PFK is also involved in reduction of the glycolytic flux during anoxia, but a more pronounced change in the MAR occurs for PK, indicating that PK is strongly inhibited under these conditions.Differences in the regulation of glycolysis in muscular and non-muscular tissues can be related to changes in metabolite levels and to tissue-specific forms of pyruvate kinase with different regulatory properties.     

Metabolic regulation during early frog development: glycogenic flux in Xenopus oocytes, eggs, and embryos

32P-labeled glucose 6-phosphate and phosphoenolpyruvate were injected into oocytes, fertilized eggs, and early embryos of Xenopus laevis, and the 32P label was followed into glycolytic enzymes and acid-soluble metabolites. The kinetics of labeling of phosphoglucomutase and phosphoglyceromutase and the formation of specific metabolites were used to measure carbon flux through glycolytic intermediates in these cells. In full-grown stage VI oocytes, fertilized eggs, and cells of cleaving embryos, carbon metabolism is in the glycogenic direction. Glycolytic intermediates injected into these cells were metabolized into UDP-glucose and then presumably into glycogen. Carbon flow between phosphoenolpyruvate and glucose 6-phosphate does not utilize fructose 1,6-bisphosphatase; rather, it may depend largely on enzymes of the pentose phosphate pathway. Maturation and fertilization of the oocyte did not result in a change in the qualitative pattern of metabolites formed. Pyruvate kinase, although abundant in oocytes and embryos, is essentially inactive in these cells. Pyruvate kinase also appears to be inactive in small previtellogenic stage II oocytes; however, in these cells injected glycolytic intermediates were not metabolized to UDP-glucose.     

Glycolytic enzymes associate dynamically with mitochondria in response to respiratory demand and support substrate channeling

In Arabidopsis thaliana, enzymes of glycolysis are present on the surface of mitochondria and free in the cytosol. The functional significance of this dual localization has now been established by demonstrating that the extent of mitochondrial association is dependent on respiration rate in both Arabidopsis cells and potato (Solanum tuberosum) tubers. Thus, inhibition of respiration with KCN led to a proportional decrease in the degree of association, whereas stimulation of respiration by uncoupling, tissue ageing, or overexpression of invertase led to increased mitochondrial association. In all treatments, the total activity of the glycolytic enzymes in the cell was unaltered, indicating that the existing pools of each enzyme repartitioned between the cytosol and the mitochondria. Isotope dilution experiments on isolated mitochondria, using (13)C nuclear magnetic resonance spectroscopy to monitor the impact of unlabeled glycolytic intermediates on the production of downstream intermediates derived from (13)C-labeled precursors, provided direct evidence for the occurrence of variable levels of substrate channeling. Pull-down experiments suggest that interaction with the outer mitochondrial membrane protein, VDAC, anchors glycolytic enzymes to the mitochondrial surface. It appears that glycolytic enzymes associate dynamically with mitochondria to support respiration and that substrate channeling restricts the use of intermediates by competing metabolic pathways.     

Effect of tranylcypromine on glycogenolysis in the rat brain

In rat brain the tranylcypromine administration induces after two hours a 151% increase of serotonin levels and a 50% fall of glycogen concentration. The activity of phosphorylase a and b and the glycolytic flow rate are unchanged.     

Down-regulation of pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) activity in sugarcane enhances sucrose accumulation in immature internodes

Pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) activity was successfully down-regulated in sugarcane using constitutively expressed antisense and untranslatable forms of the sugarcane PFP-β gene. In young internodal tissue activity was reduced by up to 70% while no residual activity could be detected in mature tissues. The transgenic plants showed no visible phenotype or significant differences in growth and development under greenhouse and field conditions. Sucrose concentrations were significantly increased in the immature internodes of the transgenic plants but not in the mature internodes. This contributed to an increase in the purity of the immature tissues, resembling an early ripening phenotype. Both the immature and mature internodes of the transgenic plants had significantly higher fibre contents. These findings suggest that PFP influences the ability of young, biosynthetically active sugarcane culm tissue to accumulate sucrose but that the equilibrium of the glycolytic intermediates, including the stored sucrose, is restored when ATP-dependent phosphofructokinase and the residual PFP activity is sufficient to sustain the required glycolytic flux as the tissue matures. Moreover, it suggests a role for PFP in glycolytic carbon flow, which could be rate limiting under conditions of high metabolic activity.     

Possible regulatory interactions between compartmentalized glycolytic systems during initiation of glycolylsis in ascites tumor cells

Changes were measured in the rates of respiration and in the levels of glycolytic intermediates during the first 5 min after addition of 1.6 mM glucose to a suspension (5%, v/v) of respiring Ehrlich ascites carcinoma cells incubated in an isotonic 50 mM tris(hydroxymethyl)methylglycine buffer (pH 7.4) at 38 °C. The rates of accumulation of lactate and glycolytic intermediates were used to calculate the in vitro velocities of glycolytic enzymes.The initial velocities of hexokinase (EC 2.7.1.1), fructose-6-phosphate kinase (EC 2.7.1.11) and lactate dehydrogenase (EC 1.1.1.27) in μmoles glucose equivalents/ ml cells per min were 14, 11 and 4, respectively. The velocities of the two kinases fell sharply to less than 5 between 5 and 10 s, while the velocity of the dehydrogenase declined gradually over the first minute. The initial burst of activity in the kinases, which lasted for about 8 s, was associated with a rapid accumulation of phosphate ester and a negative net ATP generation by glycolysis. The accumulation of phosphate ester is almost exactly matched by the generation of ATP by the “tail end” of glycolysis (triose-P to lactate) in this period. After this time (10–25 s) the rate of oxidative phosphorylation calculated as six times the rate of O₂ consumption, is nearly identical to the combined rate of ATP utilization by hexokinase and fructose-6-phosphate kinase. As observed previously, oxamate (42 mM) blocked lactate dehydrogenase but did not depress the rate of phosphate ester accumulation.These various observations and correlations can be interpreted in terms of a dual glycolytic system. The accumulation of phosphate ester during the first 8 s is attributed to the operation of a partial glycolytic system, System B, which includes only the first three or four enzymes of glycolysis, and which draws upon an ATP pool (Pool I) previously employed in assorted cytoplasmic phosphorylations. The ADP generated by System B is rephosphorylated by and regulates the rate of a complete glycolytic system A, which converts glucose to lactate with little intermediate accumulation. The tail end of System A generates a new pool of ATP (Pool II) and controls the rate of glucose input through its head end, which is supplied by ATP being produced by oxidative phosphorylation. This scheme of interlocking controls is transient and alters after 8 s, when System B slows to a stop.     

Effects of Cyanide and Ethylene on the Respiration of Cyanide-sensitive and Cyanide-resistant Plant Tissues

The effects of cyanide and ethylene, respectively, were studied on the respiration of a fully cyanide-sensitive tissue-the fresh pea, a slightly cyanide-sensitive tissue-the germinating pea seedling, and a cyanide-insensitive tissue-the cherimoya fruit. Cyanide inhibition of both fresh pea and pea seedling respiration was attended by a conventional Pasteur effect where fermentation was enhanced with an accumulation of lactate and ethanol and a change in the level of glycolytic intermediates indicative of the activation of phosphofructokinase and pyruvate kinase accompanied by a sharp decline in ATP level. In these tissues, ethylene had little or no effect on the respiration rate, or on the level of glycolytic intermediates or ATP. By contrast, ethylene as well as cyanide enhanced both respiration and aerobic glycolysis in cherimoya fruits with no buildup of lactate and ethanol and with an increase in the level of ATP. The data support the proposition that for ethylene to stimulate respiration the capacity for cyanide-resistant respiration must be present.