Ventilatory and blood gas adjustments in exercising isothermic ducks

Summary The free-living planarianPolycelis nigra has a complete sequence of glycolytic and tricarboxylic acid cycle enzymes together with an active betaoxidation sequence. Neither octopine dehydrogenase nor any other of the pyruvate: amino acid-linked dehydrogenases was present inP. nigra. The lactate dehydrogenase of this planarian was, however, unusual in being activated by fructose-1,6-bisphosphate.The steady state contents of the glycolytic and tricarboxylic acid cycle intermediates were measured in quick frozenP. nigra. A comparison of the mass action ratios with the equilibrium constants for the glycolytic reactions showed that phosphoglucomutase, glucosephosphate isomerase, aldolase, triosephosphate isomerase, phosphoglyceromutase and phosphopyruvate hydratase reactions are all near equilibrium, whilst phosphofructokinase and pyruvate kinase reactions are displaced from equilibrium. No phosphagen or phosphagen phosphotransferase activity could be detected inP. nigra but it is possible that the high levels of 3-phosphoglycerate could function as an alternative store of high energy phosphate.Under anaerobic conditionsP. nigra produces lactic acid; there is no evidence for the production of succinate, acetate or propionate, acids characteristically produced by parasitic platyhelminths.     

Cytosolic pH during a rest-to-work transition in red muscle: application of enzyme equilibria

Muscles sampled from a vascularly isolated autoperfused dog gracilis by fast freezing techniques at 5, 10, 15, 30, 60, and 180 s after the initiation of twitch contractions at 4 Hz were analyzed for phosphocreatine, creatine, ATP, lactate, pyruvate, 3-phosphoglycerate, and dihydroxyacetonephosphate contents. Metabolite concentrations were used with equilibrium constants of triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, lactate dehydrogenase, and creatine kinase to estimate cytosolic pH changes during the rest-to-work transition. Magnesium and hydrogen binding were taken into account. Limits to this approach include errors in the intermediate measurements and uncertainties in values of the equilibrium constants. The former leads to maximum errors of +/- 0.15 pH units, whereas the latter affects the absolute pH value but not estimates of the changes in pH. The estimated pH increases from a resting value of 7.05 to approximately 7.8 by 5 s of stimulation and then falls to a pH value of approximately 6.5 after 3 min of stimulation. The results are consistent with previous studies but permit identification of a larger early alkaline shift. Potential causes for the pH changes are discussed.     

Phosphocreatine production coupled to the glycolytic reactions in the cytosol of cardiac cells

Phosphocreatine production catalyzed by a cytosolic fraction from cardiac muscle containing all glycolytic enzymes and creatine kinase in a soluble form has been studied in the presence of creatine, adenine nucleotides and different glycolytic intermediates as substrates. Glycolytic depletion of glucose, fructose 1,6-bis(phosphate) and phosphoenolpyruvate to lactate was coupled to efficient phosphocreatine production. The molar ratio of phosphocreatine to lactate produced was close to 2.0 when fructose 1,6-bis(phosphate) was used as substrate and 1.0 with phosphoenolpyruvate. In these processes the creatine kinase reaction was not the rate-limiting step: the mass action ratio of the creatine kinase reaction was very close to its equilibrium value and the maximal rate of the forward creatine kinase reaction exceeded that of glycolytic flux by about 6-fold when fructose 1,6-bis(phosphate) was used as a substrate. Therefore, the creatine kinase raction was continuously in the state of quasiequilibrium and the efficient synthesis of phosphocreatine observed is a result of constant removal of ADP by the glycolytic system at an almost unchanged level of ATP ([ATP] ? [ADP]), this leading to a continuous shift of the creatine kinase equilibrium position.When phosphocreatine was added initially at concentrations of 5–15 mM the rate of the coupled creatine kinase and glycolytic reactions was very significantly inhibited due to a sharp decrease in the steady-state concentration of ADP. Therefore, under conditions of effective phosphocreatine production in heart mitochondria, which maintain a high phosphocreatine: creatine ratio in the myoplasm in vivo, the glycolytic flux may be suppressed due to limited availability of ADP restricted by the creatine kinase system. The possible physiological role of the control of the glycolytic flux by the creatine kinase system is discussed.     

Identification of the regulatory steps in glycolysis in potato tubers

The aim of this work was to identify the regulatory reactions of glycolysis in potato tubers. The amounts of glycolytic intermediates in aerobic and anoxic tubers were measured in freeze-clamped samples of tissue. Comparison of mass—action ratios with apparent equilibrium constants showed that in vivo the reactions catalysed by glucosephosphate isomerase, phosphoglycerate mutase and enolase were close to equilibrium. The ratios fructose-1,6-bisphosphate:fructose 6-phosphate, and pyruvate:phosphoenolpyruvate, respectively, showed that the reactions catalysed by phosphofructokinase and pyruvate kinase were considerably displaced from equilibrium. Stimulation of glycolysis by placing tubers in an atmosphere of nitrogen led to significant declines in their contents of fructose-6-phosphate and phosphoenolpyruvate. It is concluded that phosphofructokinase plays a dominant role in regulating entry into glycolysis, and that pyruvate kinase may regulate exit from glycolysis and the oxidative pentose phosphate pathway. Cold-induced sweetening of the tubers is discussed in the light of the above conclusions.     

Dynamics of intracellular metabolites of glycolysis and TCA cycle during cell-cycle-related oscillation in Saccharomyces cerevisiae

In the present work LC-MS/MS was applied to measure the concentrations of intermediates of glycolysis and TCA cycle during autonomous, cell-cycle synchronized oscillations in aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae. This study complements previously reported oscillations in carbon dioxide production rate, intracellular concentrations of trehalose and various free amino acids, and extracellular acetate and pyruvate in the same culture. Of the glycolytic intermediates, fructose 1,6-bisphosphate, 2- and 3-phosphoglycerate, and phosphoenolpyruvate show the most pronounced oscillatory behavior, the latter three compounds oscillating out of phase with the former. This agrees with previously observed metabolic control by phosphofructokinase and pyruvate kinase. Although individually not clearly oscillating, several intermediates of the TCA cycle, i.e., alpha-ketoglutarate, succinate, fumarate, and malate, exhibited increasing concentration during the cell cycle phase with high carbon flux through glycolysis and TCA cycle. The average mass action ratios of beta-phosphoglucomutase and fumarase agreed well with previously determined in vitro equilibrium constants. Minor differences resulted for phosphoglucose isomerase and enolase. Together with the observed close correlation of the pool sizes of the involved metabolites, this might indicate that, in vivo, these reactions are operating close to equilibrium, whereby care must be taken due to possible differences between in vivo and in vitro conditions. Combining the data with previously determined intracellular amino acid levels from the same culture, a few clear correlations between catabolism and anabolism could be identified: phosphoglycerate/serine and alpha-ketoglutarate/lysine exhibited correlated oscillatory behavior, albeit with different phase shifts. Oscillations in intracellular amino acids might therefore be, at least partly, following oscillations of their anabolic precursors.     

Combined glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase in catecholamine-stimulated guinea-pig cardiac muscle. Comparison with mass-action ratio of creatine kinase.

The steady-state reactant levels of triose-phosphate isomerase and the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system were examined in guinea-pig cardiac muscle. Key glycolytic intermediates, including glyceraldehyde 3-phosphate were directly measured and compared with those of creatine kinase. Non-working Langendorff hearts as well as isolated working hearts were perfused with 5 mM glucose (plus insulin) under normoxia conditions to maintain lactate dehydrogenase near-equilibrium. The cytosolic phosphorylation potential ([ATP]/([ADP].[Pi])) was derived from creatine kinase and the free [NAD+]/([NADH].[H+]) ratio from lactate dehydrogenase. In Langendorff hearts glycolysis was varied from near-zero flux (hyperkalemic cardiac arrest) to higher than normal flux (normal and maximum catecholamine stimulation). The triose-phosphate isomerase was near-equilibrium only in control or potassium-arrested Langendorff hearts as well as in postischemic 'stunned' hearts. However, when glycolytic flux increased due to norepinephrine or due to physiological pressure-volume work the enzyme was displaced from equilibrium. The alternative phosphorylation ratio [ATP]'/([ADP]).[Pi]) was derived from the magnesium-dependent glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system assigning free magnesium different values in the physiological range (0.1-2.0 mM). As predicted, [ATP]/([ADP].[Pi]) and [ATP]'/([ADP]'.[Pi]') were in excellent agreement when glycolysis was virtually halted by hyperkalemic arrest (flux approximately 0.2 mumol C3.min-1.g dry mass-1). However, the equality between the two phosphorylation ratios was not abolished upon resumption of spontaneous beating and also not during adrenergic stimulation (flux approximately 5-14 mumol C3.min-1.g dry mass-1). In contrast, when flux increased due to transition from no-work to physiological pressure-volume work (rate increase from approximately 3 to 11 mumol C3.min-1.g dry mass-1), the two ratios were markedly different indicating disequilibrium of the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase. Only during adrenergic stimulation or postischemic myocardial 'stunning', not due to hydraulic work load per se, glyceraldehyde-3-phosphate levels increased from about 4 microM to greater than or equal to 16 microM. Thus the guinea-pig cardiac glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system can realize the potential for near-equilibrium catalysis at significant flux provided glyceraldehyde-3-phosphate levels rise, e.g., due to 'stunning' or adrenergic hormones.     

Compartmentalized ATP synthesis in skeletal muscle triads.

Isolated skeletal muscle triads contain a compartmentalized glycolytic reaction sequence catalyzed by aldolase, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase. These enzymes express activity in the structure-associated state leading to synthesis of ATP in the triadic junction upon supply of glyceraldehyde 3-phosphate or fructose 1,6-bisphosphate. ATP formation occurs transiently and appears to be kinetically compartmentalized, i.e., the synthesized ATP is not in equilibrium with the bulk ATP. The apparent rate constants of the aldolase and the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase reaction are significantly increased when fructose 1,6-bisphosphate instead of glyceraldehyde 3-phosphate is employed as substrate. The observations suggest that fructose 1,6-bisphosphate is especially effectively channelled into the junctional gap. The amplitude of the ATP transient is decreasing with increasing free [Ca2+] in the range of 1 nM to 30 microM. In the presence of fluoride, the ATP transient is significantly enhanced and its declining phase is substantially retarded. This observation suggests utilization of endogenously synthesized ATP in part by structure associated protein kinases and phosphatases which is confirmed by the detection of phosphorylated triadic proteins after gel electrophoresis and autoradiography. Endogenous protein kinases phosphorylate proteins of apparent Mr 450,000, 180,000, 160,000, 145,000, 135,000, 90,000, 54,000, 51,000, and 20,000, respectively. Some of these phosphorylated polypeptides are in the Mr range of known phosphoproteins involved in excitation-contraction coupling of skeletal muscle, which might give a first hint at the functional importance of the sequential glycolytic reactions compartmentalized in triads.     

ATP-ADP-dependent phosphorylations of glycolysis metabolites, creatine and glycerol: their compartition and thermodynamic relationship in gastrocnemius muscle cell of exercised guinea pigs

The concentrations of following metabolites were determined in freeze-clamped gastrocnemius muscle samples: glucose 1-phosphate, glucose 6-phosphate, glucose, fructose 1,6-diphosphate, fructose 6-phosphate, D-glyceraldehyde 3-phosphate. dihydroxyacetone phosphate, phosphoenolpyruvate, pyruvate, glycerol 3-phosphate, glycerol, creatine phosphate, creatine, glycerate 3-phosphate, glycerate 2-phosphate, adenosine monophosphate, adenosine diphosphate, adenosine triphosphate, inorganic phosphate. The results showed that within the limits of experimental error, concentration homeostasis for this metabolites is founded at least in some cases on equilibria between enzymic transformations. Discrepancies between constant mass ratios measured in this study and equilibrium constants allow the free energy variation delta G to keep creatine phosphate at high concentration to be calculated. For the phosphoglycerate mutase system, the equilibrium constant in controls and trained animals is unchanged and corresponds to that in vitro. Training hindered glycolysis and favoured phosphorylation of creatine by glycerol 3-phosphate. Metabolites of the pyruvate kinase and hexokinase system cannot be homogeneously distributed in one space. The creatine kinase system is also separated from the hexokinase und pyruvate kinase system. A compartition of glycolytic process in gastrocnemius muscle seems to be inferred from these results.     

The effect of enzyme-enzyme complexes on the overall glycolytic rate in vivo.

Recent studies have demonstrated that most glycolytic enzymes can reversibly associate to form heterogeneous enzyme-enzyme (binary) complexes in vitro. However, kinetic analysis of these complexes has shown that the individual enzymes have a varied response to complex formation: some enzymes are inhibited, some are activated and some are unaffected. In order to determine the potential role of binary complexes in regulating glycolytic flux, we have mathematically calculated enzyme distributions and activities using data from in vitro binding and kinetic studies. These calculations suggest that, overall, formation of binary complexes would lower flux through phosphofructokinase and aldolase, would increase flux through glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase, and would not affect flux through triosephosphate isomerase, phosphoglycerate kinase and pyruvate kinase. The implications of these results are discussed with respect to the effect of complex formation on overall glycolytic flux and on the flux through individual enzyme loci.     

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.     

Isolation and enzymic characterization of euglena proplastids

Organelles were isolated from dark-grown Euglena gracilis Klebs by sucrose density gradient centrifugation. Plastids, identified by triosephosphate isomerase and NADP glyoxylate reductase were present at an equilibrium density of 1.24 grams per cubic centimeter clearly separated from mitochondria at an equilibrium density of 1.22 grams per cubic centimeter. Assay for choline phosphotransferase and glucose-6-phosphatase showed that endoplasmic reticulum membranes were present at a density of 1.12 grams per cubic centimeter. The plastid fraction contained phosphofructokinase, pyruvate kinase, triosephosphate isomerase and aldolase indicating the operation of a glycolytic pathway. During regreening pyruvate kinase and phosphofructokinase in the developing proplastid decreased, neither enzyme being present in the mature chloroplast. However, plastids were present in the photosynthetic cell as shown by a peak of glycolysis enzymes at an equilibrium density of 1.24 grams per cubic centimeter.     

Enzymes of glycolysis and the pentose phosphate pathway during development of the rabbit stomach worm Obeliscoides cuniculi

The activities of glycolytic and other enzymes of carbohydrate metabolism were measured in free-living and parasitic stages of the rabbit stomach worm Obeliscoides cuniculi. Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucomutase, hexokinase, glucosephosphate isomerase, phosphofructokinase, aldolase, triosephosphate isomerase, α-glycerophosphatase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, phosphoenol pyruvate carboxykinase, lactate dehydrogenase, alcohol dehydrogenase, and glucose-6-phosphatase activities were present in worms recovered 14, 20 and 190 days postinfection.The presence of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, and glucose-6-phosphatase indicates the possible function of a pentose phosphate pathway and a capacity for gluconeogenesis, respectively, in these worms.The ratio of pyruvate kinase (PK) to phosphoenol pyruvate carboxykinase (PEPCK) less than I in parasitic stages suggests that their most active pathway is that fixing CO₂ into phosphoenol pyruvate to produce oxaloacetate.Low levels of glucose-6-phosphate dehydrogenase, triosephosphate isomerase, PEPCK and PK were recorded in infective third-stage larvae stored at 5°C for 5 and 12 mos. The ratio of PK to PEPCK greater than 1 indicates that infective larvae preferentially utilize a different terminal pathway than the parasitic stages.     

Studies on the energy metabolism ofRana ridibunda erythrocytes

Summary Rana ridibunda erythrocytes have a complete sequence of glycolytic enzymes but not the tricarboxylic acid cycle enzymes.The steady state contents of the glycolytic intermediates were measured in quick frozenRana ridibunda erythrocytes. A comparison of the mass action ratios with the equilibrium constants for the glycolytic reactions showed that phosphoglucomutase, phosphoglucose isomerase, aldolase, triosephosphate isomerase, phosphoglycerate mutase and enolase reactions are all near equilibrium whilst hexokinase, phosphofructokinase and pyruvate kinase are displaced from equilibrium.The steady state contents of glycolytic intermediates, lactate, adenine nucleotides, inorganic phosphate have been measured during various periods up to 4 h of incubation of erythrocytes in the presence of glucose. In the incubation experiment glycolysis had been stimulated by the high pH-value of the medium. After 4 h of incubation 3 patterns of changes can be distinguished. One group of intermediates (glucose, glucose 6-phosphate, 2-phosphoglycerate and inorganic phosphate) in which the concentration of metabolites was lower than the zero time values. A second group of metabolites (fructose 6-phosphate, fructose 1,6-bisphosphate, phosphoenolpyruvate and AMP) in which the concentration was about the same at zero time and after 4 h of incubation. The metabolites of the third group (dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, 1,3-diphosphoglycerate, 2,3-diphosphoglycerate, 3-phosphoglycerate, pyruvate, lactate, ADP, ATP and glucose 1-phosphate) all increased their content during the 4 h of incubation in comparison to the zero time values.From the results it appears that in the amphibian erythrocyte glycolysis seems to be similar to that of mammalian erythrocytes as far its control and organisation is concerned down to the level of PEP, with the exception of the low concentration of phosphoglycerate compounds.Abbreviations 2,3DPG 2,3-diphosphoglycerate - EDTA [ethylene dinitrilo]-tetra-acetic acid - P _i inorganic phosphate - DTNB 5,5-dithio-bis-(2-nitrobenzoic acid) - PEP phosphoenolpyruvate - RBC red blood cells     

Kinetic parameters for the elimination reaction catalyzed by triosephosphate isomerase and an estimation of the reaction's physiological significance

Kinetic parameters for triosephosphate isomerase catalysis of the elimination reaction of an equilibrium mixture of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (DGAP) to form methylglyoxal and phosphate ion are reported for the enzyme from rabbit muscle. Pseudo-first-order rate constants for the disappearance of substrate (kelim) were determined for reactions at [Enzyme] much greater than [Substrate]. The second-order rate constant kEnz = 10.1 M-1 s-1 was determined from a plot of kelim against enzyme concentration. The kinetic parameters, determined from a steady-state kinetic analysis at [Substrate] much greater than [Enzyme], are kcat = 0.011 s-1, Km = 0.76 mM, and kcat/Km = 14 M-1 s-1. The estimated rate-constant ratio for partitioning of the enzyme-bound intermediate between protonation at carbon 2 and elimination, 1,000,000, is much larger than the ratio of 6.5 determined for the reaction of the enediolate phosphate in a loose complex with quinuclidinonium cation, a small buffer catalyst. There is a 10(5)-10(8)-fold decrease in the rate constant for the elimination reaction of the enediolate phosphate when this species binds to triosephosphate isomerase. The kinetic parameters for the elimination reaction catalyzed by the native triosephosphate isomerase and for the reaction catalyzed by a mutant form of the enzyme, which is missing a segment that forms hydrogen bonds with the phosphate group of substrate [Pompliano, D. L., Peyman, A., & Knowles, J. R. (1990) Biochemistry 29, 3186-3194] are similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Control of glycolysis in cerebral cortex slices

1. Intracellular concentrations of intermediates and cofactors of glycolysis were measured in guinea-pig cerebral cortex slices incubated under varying conditions. 2. Comparison of mass-action ratios with apparent equilibrium constants for the reactions of glycolysis showed that hexokinase, phosphofructokinase and pyruvate kinase catalyse reactions generally far from equilibrium, whereas phosphoglucose isomerase, aldolase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, adenlyate kinase and creatine phosphokinase are generally close to equilibrium. The possibility that glyceraldehyde 3-phosphate dehydrogenase may catalyse a ;non-equilibrium' reaction is discussed. 3. Correlation of changes in concentrations of substrates for enzymes catalysing ;non-equilibrium' reactions with changes in rates of glycolysis caused by alteration of the conditions of incubation showed that hexokinase, phosphofructokinase, pyruvate kinase and possibly glyceraldehyde 3-phosphate dehydrogenase are subject to metabolic control in cerebral cortex slices. 4. It is suggested that the glycolysis is controlled by two regulatory systems, the hexokinase-phosphofructokinase system and the glyceraldehyde 3-phosphate dehydrogenase-pyruvate kinase system. These are discussed. 5. It is concluded that the rate of glycolysis in guinea-pig cerebral cortex slices is limited either by the rate of glucose entry into the slices or by the hexokinase-phosphofructokinase system. 6. It is concluded that addition of 0.1mm-ouabain to guinea-pig cerebral cortex slices causes inhibition of either glyceraldehyde 3-phosphate dehydrogenase or phosphoglycerate kinase or both, in a manner independent of the known action of ouabain on the sodium- and potassium-activated adenosine triphosphatase.     

Glycolytic enzymes of Trypanosoma brucei. Simultaneous purification, intraglycosomal concentrations and physical properties

We have developed a method for the simultaneous purification of hexokinase, glucosephosphate isomerase, phosphofructokinase, fructose-1,6-bisphosphate aldolase, triosephosphate isomerase, D-glyceraldehyde-phosphate dehydrogenase, phosphoglycerate kinase, glycerol-3-phosphate dehydrogenase and glycerol kinase from Trypanosoma brucei in yields varying over 8-55%. Crude glycosomes were prepared by differential centrifugation of cell homogenates. Subsequent hydrophobic interaction chromatography on phenyl-Sepharose resulted in six pools containing various mixtures of enzymes. These pools were processed via affinity chromatography (immobilized ATP), hydrophobic interaction chromatography (octyl-Sepharose) and ion-exchange chromatography (CM- and DEAE-cellulose) which resulted in the purification of all nine enzymes. The native enzyme and subunit molecular masses, as determined by gel filtration and gel electrophoresis under denaturing conditions, were compared with those of their homologous counterparts from other organisms. Trypanosomal hexokinase is a hexamer and differs in subunit composition from the mammalian enzymes (monomers) as well as in subunit size (51 kDa versus 96-100 kDa, respectively). Phosphofructokinase only differs in subunit size (51 kDa for T. brucei versus 80-90 kDa for mammals) but had identical subunit composition (tetrameric). The others all have the same subunit composition as their mammalian counterparts. Except for triosephosphate isomerase, all Trypanosoma enzymes have subunits which are 1-5 kDa larger in size. Together these nine enzymes contribute 3.3 +/- 1.6% to the total cellular protein of T. brucei and at least 90% to the total glycosomal protein. A comparison of calculated intraglycosomal concentrations of the enzymes with the glycosomal metabolite concentrations shows that in the case of aldolase, glyceraldehyde-phosphate dehydrogenase and phosphoglycerate kinase, the concentration of active sites is of the same order of magnitude as that of their reactants. A common feature of the glycosomal glycolytic enzymes (with the exception of glucosephosphate isomerase) is that they are highly basic proteins with pI values between 8.8 and 10.2, values which are 1-4 higher than in the case of their mammalian cytosolic counterparts and 3-6 higher than in the case of the various unicellular organisms. It is suggested that both the larger subunit size and the basic character of the T. brucei glycolytic proteins are involved in the routing of the enzymes from their site of biogenesis (the cytosol) towards their site of action (the glycosome).     

The role of the mitochondrial creatine kinase system for myocardial function during ischemia and reperfusion.

The subcellular distribution of ATP, ADP, creatine phosphate and creatine was studied in normoxic control, isoprenaline-stimulated and potassium-arrested guinea-pig hearts as well as during ischemia and after reperfusion. The mitochondrial creatine phosphate/creatine ratio was closely correlated to the oxidative activity of the hearts. This was interpreted as an indication of a close coupling of mitochondrial creatine kinase to oxidative phosphorylation. To further investigate the functional coupling of mitochondrial creatine kinase to oxidative phosphorylation, rat or guinea-pig heart mitochondria were isolated and the mass action ratio of creatine kinase determined at active or inhibited oxidative phosphorylation or in the presence of high phosphate, conditions which are known to change the functional state of the mitochondrial enzyme. At active oxidative phosphorylation the mass action ratio was one-third of the equilibrium value whereas at inhibited oxidative phosphorylation (N2, oligomycin, carboxyatractyloside) or in the presence of high phosphate, the mass action ratio reached equilibrium values. These findings show that oxidative phosphorylation is essential for the regulation of the functional state of mitochondrial creatine kinase. The functional coupling of the mitochondrial creatine kinase and oxidative phosphorylation indicated from the correlation of mitochondrial creatine phosphate/creatine ratios with the oxidative activity of the heart in situ as well as from the deviation of the mass action ratio of the mitochondrial enzyme from creatine kinase equilibrium at active oxidative phosphorylation in isolated mitochondria is in accordance with the proposed operation of a creatine shuttle in heart tissue.     

Glycolysis in Entamoeba histolytica. Biochemical characterization of recombinant glycolytic enzymes and flux control analysis

The synthesis of ATP in the human parasite Entamoeba histolytica is carried out solely by the glycolytic pathway. Little kinetic and structural information is available for most of the pathway enzymes. We report here the gene cloning, overexpression and purification of hexokinase, hexose-6-phosphate isomerase, inorganic pyrophosphate-dependent phosphofructokinase, fructose-1,6 bisphosphate aldolase (ALDO), triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase, phosphoglycerate mutase (PGAM), enolase, and pyruvate phosphate dikinase (PPDK) enzymes from E. histolytica. Kinetic characterization of these 10 recombinant enzymes was made, establishing the kinetic constants at optimal and physiological pH values, analyzing the effect of activators and inhibitors, and investigating the storage stability and oligomeric state. Determination of the catalytic efficiencies at the pH optimum and at pH values that resemble those of the amoebal trophozoites was performed for each enzyme to identify possible controlling steps. This analysis suggested that PGAM, ALDO, GAPDH, and PPDK might be flux control steps, as they showed the lowest catalytic efficiencies. An in vitro reconstruction of the final stages of glycolysis was made to determine their flux control coefficients. Our results indicate that PGAM and PPDK exhibit high control coefficient values at physiological pH.     

The influence of fructose-1:6-bisphosphate on the release of glycolytic enzymes from cellular structure

In order to provide information on the relative binding characteristics of glycolytic enzymes, the effect of fructose-1,6-bisphosphate (FBP) on the release of glycolytic enzymes from cultured pig kidney cells treated with digitonin has been studied. In the absence of FBP, a differential release of these enzymes was observed, with the order of retention being aldolase greater than glyceraldehyde-3-phosphate dehydrogenase greater than glucosephosphate isomerase, triosephosphate isomerase, phosphoglycerokinase, phosphoglucomutase, lactate dehydrogenase, enolase, pyruvate kinase and phosphofructokinase. In the presence of fructose-1,6-bisphosphate, the release of aldolase was considerably enhanced, whereas the release of phosphofructokinase and pyruvate kinase was decreased by this metabolite. No significant alterations in the rate of release of the other enzymes was caused by FBP. These data have been discussed in relation to their contribution to the knowledge of the degree of association and order of binding between glycolytic enzymes and the cytoplasmic matrix.