Metabolite Concentrations and Phase Relations in d-Fructose Induced Glycolytic Oscillations

The frequency of glycolytic oscillations in yeast cells is only slightly modified by the consumption rate of either glucose or fructose, but it is reduced to 1 /2 to 2/3 if the ketohexose is the only substrate. Phase relations between NADH, adenine nucleotides and sugar phosphates (G-6-P, F-6-P, FDP, DAP) are independent of the hexose fermented. With fructose as a substrate the amplitudes of adenylates and hexose phosphates are distinctly smaller than with glucose. The maximum of G-6-P is higher with glucose and the minimum concentration of FDP is higher with fructose. In the transition to anaerobiosis FDP, adenosine 5′-diphosphate and adenosine 5′-phosphate are extremely high, whereas G-6-P, F-6-P and ATP concentrations are lower if fructose is the substrate fermented. The results are indicative for different control characteristics of the phosphofructokinase step, but on their basis it cannot be distinguished between a direct interaction of fructose (or one of its derivatives) on the phosphofructokinase kinetics or the existence of a bypass for fructose which might be able to withdraw a part of the substrate from the control point at the phosphofructokinase.     

Catabolism of d-fructose and d-ribose by Pseudomonas doudoroffii

1. The 1-P-fructokinase (1-PFK) and 6-P-fructokinase (6-PFK) from Pseudomonas doudoroffii were partially purified by a combination of (NH₄)₂SO₄ fractionation and DEAE-Sephadex column chromatography. The pH optima of these enzymes were 9.0 and 8.5, respectively.
2. When the concentrations of the substrates of the 1-PFK reaction were varied, Michaelis-Menten kinetics were observed. The Kms for d-fructose-1-P (F-1-P) and ATP were 3.03×10^-4 M and 3.39×10^-4 M, respectively. Variation of MgCl₂ at fixed concentrations of F-1-P and ATP resulted in sigmoidal kinetics; about 10 mM MgCl₂ was necessary for maximal activity. Activity of 1-PFK was inhibited when the ratio of ATP: Mg⁺⁺ was higher than 0.5, suggesting that ATP: 2Mg⁺⁺ was the substrate and that free ATP was inhibitory. Although an absolute requirement for K⁺ or NH ₊ ⁴ could not be demonstrated, these cations stimulated the rate of the reaction. Activity of 1-PFK was not significantly affected by 3 mM AMP, cyclic-AMP, P_i, d-fructose-6-P (F-6-P), ADP, P-enolpyruvate (PEP), pyruvate, citrate, or l-glutamate.
3. Sigmoidal kinetics were observed for 6-PFK when the concentration of F-6-P was increased and the level of ATP was kept constant. Activity of 6-PFK was increased by ADP, inhibited by PEP, and unaffected by 3 mM AMP, cyclic-AMP, P_i, F-1-P, pyruvate, or citrate.

     

Cytosolic phosphofructokinase from spinach leaves : I. Purification, characteristics, and regulation

Cytosolic ATP-dependent phosphofructokinase (PFK) from spinach leaves (Spinacia oleracea L.) was enriched 2600-fold by (NH₄)₂SO₄ fractionation, DEAE anion exchange chromatography, Blue Sepharose CL-6B, and ATP agarose type 3-affinity chromatography. The final preparation had a specific activity of 417 nkat per milligram protein and exhibited four bands between 50 and 70 kilodaltons following denaturing electrophoresis. Only one band of ATP- and fructose 6-phosphate (F-6-P)-dependent, Pistimulated activity was detected following isoelectric focusing PAGE and nondenaturing discontinuous PAGE of the final preparation. Crude extracts contained, in addition to the band observed in the final preparation, a second band that was inhibited by Pi. The latter band is presumably chloroplastic PFK. PFK was stimulated by the anions Pi²⁻, Cl⁻, SO₄²⁻, NO₃⁻, HAsO₄²⁻, and HCO₃⁻ but was not affected by NH₄⁺. Pi and Mg²⁺ changed the response of PFK toward pH and affected the saturation kinetics of F-6-P. In general, activity was highest when Pi was high and (or) Mg²⁺ was low. Phosphoenolpyruvate (PEP), 2-PGA, and PPi, but not 3-PGA, inhibited PFK. Although the inhibition by PEP and 2-PGA was reduced or relieved by Pi, the inhibition by PPi was not affected by Pi. F-2, 6-P₂ had no effect upon the activity of PFK. It is proposed that, in the cytosol of spinach leaves, PFK is likely to be more active during the dark, when cytosolic Pi levels are high, than in the light.     

Interactions between magnesium ions,pH, glucose-6-phosphate,and NADPH/NADP+ ratios in the modulation of chloroplast glucose-6-phosphate dehydrogenase in vitro

Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from spinach chloroplasts is strongly affected by interactions between Mg²⁺, proton, and substrate concentrations. Mg²⁺ activates the enzyme to different degrees; however, it is not essential for enzyme activity. The Mg²⁺-dependent activation follows a maximum curve, magnitude and position of the maximum being dependent on pH and NADPH/NADP⁺ ratios. At a ratio of zero and pH 7.2, maximum activity is observed at 10 mM Mg²⁺. Increasing the NADPH/NADP⁺ ratio up to 1.7 (a ratio measured in the stroma during a light period), maximum activity is shifted to much lower Mg²⁺ concentrations. At pH 8.2 (corresponding to the pH of the stroma in the light) and at a high NADPH/NADP⁺ ratio, enzyme activity is not affected by the Mg²⁺ ion. The results are discussed in relation to dark-light-dark regulation of the oxidative pentose phosphate cycle in spinach chloroplasts.Abbreviations DTT dithiothreitol - G-6-P glucose-6-phosphate - G-6-PDH glucose-6-phosphate dehydrogenase (EC 1.1.1.49) - PPC pentose phosphate cycle     

Effect of magnesium and ATP on ATPase of sugarcane vacuoles

Kinetic analysis of the Mg²⁺-dependence of tonoplast ATPase from suspension-cultured cells of sugarcane showed that the enzyme activity increased with increasing magnesium concentrations till 1–3 mM and then decreased consideably for higher concentrations. This kinetic could be explained by the assumption that MgATP^2- is the substrate of ATPase: MgATP^2- concentration increases with increasing concentration of magnesium till, at high concentrations of magnesium, Mg₂ATP is formed. No evidence for a direct role of Mg²⁺ as activator or inhibitor was found. These data corroborate previous findings that MgATP^2- is the sole substrate of the vacuolar ATPase of sugarcane (Thom and Komor 1984). High concentrations of ATP seemed to inhibit the ATPase. This result, however, could be traced back to interference of ATP with the Fiske-Subbarow method of phosphate determination. After adjustment of the test conditions, inhibition by ATP was no longer found. Reported data for ATPases of other plant materials, showing inhibition of enzyme activity with high magnesium or ATP concentrations, might be explicable in a similar way.Abbreviation Mes 2-(N-morpholino)ethane+Sulfonic acid     

Purification and characterization of phosphofructokinase of Bacillus licheniformis

The phosphofructokinase (PFK) of Bacillus licheniformis was purified about 50–65-fold and examined for a number of enzymatic and physical characteristics. The enzyme is quite unstable under normal assay conditions, but Mg²⁺, K⁺, adenosine-5′-diphosphate, phosphoenolpyruvate (PEP), and fructose-6-phosphate (fru-6-P) are fairly effective stabilizing agents. Saturation functions for ATP and fru-6-P were hyperbolic. Several attempts to induce positive cooperative binding of fru-6-P were unsuccessful. However, “sigmoidal” saturation kinetics for fru-6-P could be observed under assay conditions that permitted an irreversible inactivation of the PFK during assay. Several divalent cations could support the catalysis of B. licheniformis PFK and the enzyme was activated by both NH₄⁺ and K⁺ ions. B. licheniformis PFK is inhibited by citrate, ATP, PEP, Ca²⁺, and several other metabolic intermediates, but the inhibition caused by citrate and ATP at high fru-6-P concentration and by calcium can be relieved by Mg²⁺ addition while PEP inhibition is specifically relieved by fru-6-P. There are at least three binding sites for PEP on the PFK molecule. The active form of this PFK has a molecular weight of about 134,000 daltons. In the presence of Mg²⁺, adenosine-5′-triphosphate (ATP), and PEP, at 0 °C, the PFK molecule is rapidly dissociated to an inactive form with a molecular weight of about 68,000 daltons. Association of these subunits to yield the active form of PFK occurs spontaneously, and rapidly, when the temperature is raised to 30 °C. Ninety percent of the original activity is recovered after activation. Growth of B. licheniformis on several different substrates resulted in minor variations of PFK activity. In a parallel fashion, sporulation involved no irreversible inactivation of PFK and the level of the activity was about the same throughout the life cycle. Control of this enzyme during sporulation could be affected by any or all of the cell constituents found to regulate PFK activity in vitro, but it is considered likely that the most significant in vivo negative effector is PEP, with this inhibition being reversed by fru-6-P.     

The role of Mg2+ and Ca2+ in the simultaneous binding of vanadate and ATP at the phosphorylation site of sarcoplasmic reticulum Ca2+-ATPase

The sarcoplasmic reticulum Ca²⁺-ATPase was reacted with vanadate in the presence of Mg²⁺ and EGTA, and the effect of Ca²⁺, Mg²⁺ and ATP on the kinetics of vanadate release from the enzyme vanadate complex was studied after dilution with vanadate-free media. Ca²⁺ increased, whereas ATP decreased the rate of vanadate release. In absence of free Mg²⁺ in the release media ATP was bound to the vanadate-reacted Ca²⁺-ATPase with high affinity (K_d 4–5 μM), and full saturation with ATP resulted in complete inhibition of vanadate release. In media containing free Mg²⁺, where ATP predominantly was present as MgATP, binding of the nucleotide to vanadate-reacted Ca²⁺-ATPase occurred with low apparent affinity. Mg²⁺ alone did not affect the rate of vanadate release. At saturating ATP concentrations the release rate in the presence of free Mg²⁺ was less inhibited than in its absence. These results indicate that uncomplexed ATP interacts with the same Mg²⁺ at the catalytic site, which is involved in formation of the enzyme-vanadate complex (EMgV), and thereby hinders dissociation of vanadate. Destabilization of the complex by free Mg²⁺ may be caused by the presence of an additional magnesium ion in the catalytic site together with ATP.     

Interaction of inhibitors with muscle phosphofructokinase.

The interaction of several inhibitors with muscle phosphofructokinase has been studied by both equilibrium binding measurements and kinetic analysis. At low concentrations of citrate a maximum of 1 mol is bound per mol of enzyme protomer. Tight binding requires MgATP and very weak binding is observed in the absence of either magnesium ion or ATP. ITP at low concentrations cannot replace ATP. In the presence of MgATP and at pH 7.0, the dissociation constant for the enzyme-citrate complex is 20 muM. At 50 muM citrate and excess magnesium ion, the concentration of ATP required to give half-maximal binding of citrate is approximately 3 muM . Both P-enolpyruvate and 3-P-glycerate compete for the binding of citrate and the estimated Ki values are 480 and 52 muM, respectively. Creatine-P, another inhibitor of muscle phosphofructokinase, does not compete with the binding of citrate. Measurement of the equilibrium binding of ATP shows that citrate, 3-P-glycerate, P-enolpyruvate, and creatine-P all increase the affinity of enzyme for MgATP with the concentration required to give an effect increasing in the order given. In kinetic studies, citrate, 3-P-glycerate and P-enolpyruvate each act synergistically with ATP to inhibit the phosphofructokinase reaction. This is indicated by the observation that the three metabolites do not inhibit the enzyme with ITP as the phosphoryl donor and that they inhibit at ATP concentrations that are not themselves inhibitory. Furthermore, the sensitivity to the inhibitors increases with increasing ATP concentrations. Striking differences in the extent of inhibition can be seen by varying the order of addition of assay components. Preincubation of the enzyme with ATP and citrate, 3-P-glycerate, or P-enolpyruvate results in greater inhibition than when the inhibitor is added after the reaction is started with fructose-6-P. Furthermore, the inhibition is reversed partially 10 to 15 min after the addition of fructose-6-P. This phenomenon is particularly striking with creatine-P as the inhibitor. Very high concentrations of this inhibitor are required to show any effect if the inhibitor is added after fructose-6-P. These effects are interpreted as reflecting slow conformational changes between an active form with high affinity for fructose-6-P and an inactive, or less active, conformation that binds the inhibitors. Citrate, 3-P-glycerate, P-enolpyruvate, and creatine-P increase the rate of the phosphofructokinase at subsaturating concentrations of MgITP. The results indicate a common binding site on the enzyme for citrate, 3-P-glycerate, and P-enolpyruvate that is distinct from the ATP inhibitory site. An additional site (or sites) for creatine-P is indicated. All four inhibitors act synergistically with ATP by increasing the affinity of the enzyme for MgATP at an inhibitory site. The inhibitors appear also to increase the affinity of the catalytic nucleoside triphosphate site for substrate.     

Oscillatory synthesis of glucose 1,6-bisphosphate and frequency modulation of glycolytic oscillations in skeletal muscle extracts

Oscillatory behavior of glycolysis in cell-free extracts of rat skeletal muscle involves bursts of phosphofructokinase activity, due to autocatalytic activation by fructose-1,6-P2. Glucose-1,6-P2 similarly might activate phosphofructokinase in an autocatalytic manner, because it is produced in a side reaction of phosphofructokinase and in a side reaction of phosphoglucomutase using fructose-1,6-P2. When muscle extracts were provided with 1 mM ATP and 10 mM glucose, glucose-1,6-P2 accumulated in a stepwise, but monotonic, manner to 0.7 microM in 1 h. The stepwise increases occurred during the phases when fructose-1,6-P2 was available, consistent with glucose-1,6-P2 synthesis in the phosphoglucomutase side reaction. Addition of 5-20 microM glucose-1,6-P2 increased the frequency of the oscillations in a dose-dependent manner and progressively shortened the time interval before the first burst of phosphofructokinase activity. Addition of 30 microM glucose-1,6-P2 blocked the oscillations. The peak values of the [ATP]/[ADP] ratio were then eliminated, and the average [ATP]/[ADP] ratio was reduced by half. In the presence of higher, near physiological concentrations of ATP and citrate (which reduce the activation of phosphofructokinase by glucose-1,6-P2), high physiological concentrations of glucose-1,6-P2 (50-100 microM) increased the frequency of the oscillations and did not block them. We conclude that autocatalytic activation of phosphofructokinase by fructose-1,6-P2, but not by glucose-1,6-P2, is the mechanism generating the oscillations in muscle extracts. Glucose-1,6-P2 may nevertheless play a role in facilitating the initiation of the oscillations and in modulating their frequency.     

Regulation of glycolysis and level of the Crassulacean acid metabolism

Glycolysis shows different patterns of operation and different control steps, depending on whether the level of Crassulacean acid metabolism (CAM) is low or high in the leaves of Kalanchoe blossfeldiana v.Poelln., when subjected to appropriate photoperiodic treatments: at a low level of CAM operation all the enzymes of glycolysis and phosphoenol pyruvate (PEP) carboxylase present a 12 h rhythm of capacity, resulting from the superposition of two 24h rhythms out of phase; phosphofructokinase appears to be the main regulation step; attainment of high CAM level involves (1) an increase in the peak of capacity occurring during the night of all the glycolytic enzymes, thus achieving an over-all 24h rhythm, in strict allometric coherence with the increase in PEP carboxylase capacity, (2) the establishment of different phase relationships between the rhythms of enzyme capacity, and (3) the control of three enzymic steps (phosphofructokinase, the group 3-P-glyceraldehyde dehydrogenase — 3-P-glycerate kinase, and PEP carboxylase). Results show that the hypothesis of allosteric regulation of phosphofructokinase (by PEP) and PEP carboxylase (by malate and glucose-6-P) cannot provide a complete explanation for the temporal organization of glycolysis and that changes in the phase relationships between the rhythms of enzyme capacity along the pathway and a strict correlation between the level of PEP carboxylase capacity and the levels of capacity of the glycolytic enzymes are important components of the regulation of glycolysis in relation to CAM.Abbreviations CAM crassulacean acid metabolism - F-6-P fructose-6-phosphate - F-bi-P fructose-1,6 biphosphate - G-3-PDH 3-phosphoglyceraldehyde dehydrogenase (NAD), EC 1.2.1.12 - G-6-P glucose-6-phosphate - GSH reduced glutathion - GDH glycerolphosphate dehydrogenase, EC 1.1.1.8 - PEP phosphoenol pyruvate - PEPC PEP carboxylase, EC 4.1.1.31 - PFK phosphofructokinase, EC 2.7.1.11 - 2-PGA 2-phosphoglycerate - 3-PGA 3-phosphoglycerate - PGM phosphoglycerate phosphomutase, EC 5.4.2.1 - T.P. triose phosphates - TPI triose phosphate isomerase, EC 5.3.1.1     

Activators of the ATP-dependent surface reaction in the apical cell membrane of the bull-sperm head, causing head-to-head association

Mg²⁺, Ca²⁺ and Mn²⁺ were found to act as activators of the ATP-dependent surface reaction, leading to head-to-head association in bull spermatozoa. Ca²⁺ was more efficient than Mg²⁺, while Zn²⁺, like Na⁺ + K⁺ in combination with Mg²⁺, seemed to have no such effect. High ionic strength induced head-to-head association, as did higher concentrations of Mg²⁺ and Ca²⁺ than those necessary for the activation of ATP, Ca²⁺ acting in a lower conc. than Mg²⁺. To this effect was added that of the ATP-dependent reaction when ATP was also present. As activators, Mg²⁺ and Ca²⁺ did not potentiate each other; their effects were cumulative when the ions acted together.When the ATP concentration within the range 1 × 10⁻⁵ to 8 × 10⁻⁵ M was increased stepwise in the presence of 2 × 10⁻⁵ M Mg²⁺ or Ca²⁺, the association resulting from each single concentration step progressively increased. At low cation concentrations, the increase was about the same for the two cations: at higher concentrations it was much steeper in the presence of Ca²⁺ than in that of Mg²⁺. In the latter case, it was not statistically significant above 4 × 10⁻⁵ M ATP.Increasing the cation concentration in the range 1 × 10⁻⁵ to 4 × 10⁻⁵ M in the presence of 2 × 10⁻⁵ M ATP produced an immediate high increase in association, which was followed by a lower increase. The optimum concentration ratio for Mg²⁺:ATP was at least 1:1 and for Ca²⁺: ATP at least 1.5:1.Oubain, containing enone structure, abolishes association.     

Calcium,magnesium and the control of PTH secretion

Critical to the regulation of mineral ion homeostasis is the inverse relationship bewtween the extracellular calcium (Ca²⁺) concentration and PTH secretion (Fig. 1). Early studies in vivo [1] and in vitro [2] demonstrated that high magnesium (Mg²⁺) concentrations also inhibit PTH release. At low concentrations, on the other hand, the effects of Ca²⁺ and Mg²⁺ on PTH secretion differ: while hormonal secretion persists for an hour or more even at vanishingly low Ca²⁺ concentrations [3,4], Connie Anast and his co-workers were the first to demonstrate that low Mg²⁺ concentrations inhibit PTH secretion [5]. In these classical studies, Dr Anast was able to use detailed clinical observations in a single patient to draw important pathophysiological conclusions. Indeed, in the ensuing years, only limited progress has been made in extending these observations to elucidate the cellular mechanisms underlying the inhibition of PTH release at low Mg²⁺. Considerable advances, on the other hand, have been made in understanding the control of PTH release by high Ca²⁺ and Mg²⁺ concentrations. These studies will be reviewed here and provide, in turn, a conceptual framework within which to consider the effects of low Mg²⁺ on PTH secretion.     

Effects of insulin and work on fructose 2,6-bisphosphate content and phosphofructokinase activity in perfused rat hearts

The effects of insulin and increased cardiac work on glycolytic rate, metabolite content, and fructose 2,6-bisphosphate (Fru-2,6-P2) content were studied in isolated perfused rat hearts. Steady-state rates of glycolysis increased 5-fold with the addition of insulin to the perfusate or by increasing cardiac pressure-volume work and correlated well in most conditions with changes in substrate concentration (Fru-6-P) and with concentration of the activator, Fru-2,6-P2. There was no correlation with changes in other well known regulators including citrate, ATP, AMP, Pi, or cytosolic phosphorylation potential. Using phosphofructokinase purified from hearts perfused under identical conditions, allosteric kinetic experiments were performed using the metabolite and effector concentrations determined from in vivo experiments. Reaction rates for phosphofructokinase calculated in vitro agreed well with the glycolytic rates measured in vivo and correlated with changes in Fru-6-P but not with other effectors. However, higher Fru-2,6-P2 levels were more effective in maintaining phosphofructokinase activity at high ATP and citrate levels. Kinetic experiments did not indicate a covalent modification of phosphofructokinase. These data indicate that control of cardiac phosphofructokinase and glycolysis may be accomplished by changes in the availability of substrate, Fru-6-P, and activator, Fru-2,6-P2, rather than by citrate, adenine nucleotides, or cytosolic phosphorylation potential as previously suggested.     

Modulation of muscle phosphofructokinase at physiological concentration of enzyme

Two approaches have been used to study the allosteric modulation of phosphofructokinase at physiological concentration of enzyme; a "slow motion" approach based on the use of a very low Mg2+/ATP ratio to conveniently lower Vmax, and the addition of polyethylene glycol as a "crowding" agent to favor aggregation of diluted enzyme. At 0.6 mg/ml muscle phosphofructokinase exhibited a drastic decrease in the ATP inhibition and the concomitant increase in the apparent affinity for fructose-6-P, as compared to a 100-fold diluted enzyme. Similar results were obtained with diluted enzyme in the presence of 10% polyethylene glycol (Mr = 6000). Results with these two approaches in vitro were essentially similar to those previously observed in situ (Aragón, J. J., Felíu, F. E., Frenkel, R., and Sols, A. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 6324-6328), indicating that the enzyme is strongly dependent on homologous interactions at physiological concentrations. With polyethylene glycol it was observed that within the physiological range of concentration of substrates and the other positive effectors, fructose-2,6-P2 still activates the liver phosphofructokinase although it no longer significantly affects the muscle isozyme. In the presence of polyethylene glycol, muscle phosphofructokinase can approach its maximal rate even in the presence of physiologically high concentrations of ATP. Three minor activities of muscle phosphofructokinase have been studied at high enzyme concentration: the hydrolysis of MgATP (ATPase) and fructose-1,6-P2 (FBPase), produced in the absence of the other substrate, and the reverse reaction from MgADP and fructose-1,6-P2. The kinetic study of these activities has allowed a new insight into the mechanisms involved in the modulation of phosphofructokinase activity. The binding of (Mg)ATP at its regulatory site reduces the ability of the enzyme to cleave the bond of the terminal phosphate of MgATP at the substrate site. The positive effectors (Pi, cAMP, NH+4, fructose-1,6-P2, and fructose-2,6-P2) decrease the inhibitory effect of MgATP. Citrate and fructose-2,6-P2 both act as mechanistically "secondary" effectors in the sense that citrate does not inhibit and fructose-2,6-P2 does not activate the FBPase activity, requiring both the presence of ATP to affect the enzyme activity. In conclusion it appears that the regulatory behavior of mammalian phosphofructokinases is utterly dependent on the fact of their high concentrations in vivo.     

Chloroplast phosphofructokinase in the green alga, Dunaliella marina: partial purification and kinetic and regulatory properties

The aim of this work was to study the pathway(s) of sugar phosphate metabolism in chloroplasts of the unicellular green alga, Dunaliella marina (Volvocales). Phosphofructokinase, detectable in crude cell extracts, copurifled with intact chloroplasts on sucrose density gradients. In isolated chloroplasts, phosphofructokinase activity displayed latency to the same degree as chloroplast marker enzymes. From the quantitative distribution of enzyme activities in fractionated cells, it is concluded that there is an exclusive localization of phosphofructokinase in chloroplasts. In addition, no separation into multiple forms could be achieved. For the study of regulatory properties, chloroplast phosphofructokinase was partially purified by ammonium sulfate fractionation followed by DEAE-cellulose chromatography. The pH optimum of the enzyme activity was 7.0 and was not altered with varying concentrations of substrates or low-molecular-weight effectors. Fructose 6-phosphate showed a sigmoidal saturation curve whose shape was further changed with varying protein concentrations of the preparation. The second substrate, ATP, gave a hyperbolic saturation curve with a Michaelis constant of 60 μm. At a Mg²⁺ concentration of 2.5 mm, ATP concentrations exceeding 1 mm inhibited the enzyme in a positive cooperative manner. The same type of inhibition was observed with other phosphorylated intermediates of carbon metabolism, the most efficient being phosphoenolpyruvate, glycolate 2-phosphate, glycerate 3-phosphate, and glycerate 2-phosphate. Inorganic phosphate was the only activator found for phosphofructokinase. With nonsaturating fructose 6-phosphate concentrations, P_i activated in a positive cooperative fashion, while no activation occurred with saturating fructose 6-phosphate concentrations. In the presence of either an activator or an inhibitor, the sigmoidal shape of the fructose 6-phosphate saturation curve was altered. Most notably, the activator P_i could relieve the inhibitory action of ATP, phosphoenolpyruvate, glycerate 3-phosphate, glycerate 2-phosphate, and glycolate 2-phosphate. Based on these experimental findings, the regulatory properties of D. marina chloroplast phosphofructokinase are discussed with respect to its playing a key role in the regulation of chloroplast starch metabolism during a light/dark transition. All available evidence is compatible with the interpretation that phosphofructokinase is active only in the dark thus channeling starch degradation products into glycolysis.     

Pressure Effects on Catalysis and Control of Catalysis by Liver Fructose Diphosphatase from an Off-Shore Benthic Fish

SYNOPSIS: At low temperature (2°C), in the absence of FDPand Mg²⁺, the enzyme fructose disphosphatase (FDPase), extractedfrom the liver of an off-shore benthic Coryphaenoides species,is inactivated by exposures to relatively low pressures. Thesubstrate, FDP, and the cofactor, Mg²⁺, protect against thisinactivation, so that catalysis per se is not retarded by pressure.In contrast, at alkaline pH, pressure dramatically acceleratesthe catalytic rate when FDP and Mg²⁺ are saturating. The volumechange of activation, V*, for Coryphaenoides FDPase under theseconditions is about –40 cm³/mole. At low concentrationsof FDP and saturating concentrations of cofactor, the reactionrate at alkaline pH is pressure-independent. Similarly, at lowconcentrations of Mg²⁺ but saturating concentration of FDP,the reaction rate is pressure-independent. The K_m for FDP doesnot change measureably with pressure, while the K_a for Mg²⁺increases slightly with pressure. Under conditions of low (probablephysiological) FDP and Mg²⁺ concentrations, it is evident thatthe reaction rate is determined by the kinetic characteristicsof the enzyme and not by its energy-volume relationships, asituation which would appear to be of functional and selectivesignificance to an organism living under constantly high hydrostaticpressure. AMP is a potent specific inhibitor of CoryphaenoidesFDPase. The K₄ for AMP is essentially pressure-independent bothat neutral and alkaline pH, suggesting that efficiency of AMPcontrol of this enzyme is comparable at all pressures likelyto be encountered in nature.     

Partial purification and characterization of a novel Mg- dependent ATPase present in the cytosol from human erthrocytes

Mg²⁺-ATPase activity was identified in the cytosol of human erythrocytes. A partial purification of this activity was achieved by an initial DEAE-Sephadex column chromatography, followed by gel filtration on Sephadex G-100 and then a second DEAE-Sephadex chromatography procedure. The enzyme appeared in the void volume of the Sephadex G-100 column and was retained on an Amicon XM100A ultrafiltration membrane. The molecular weight of the enzyme was estimated to be 113 000 from SDS gels. The above purification protocol yielded an enzyme with an optimal pH between 7.6 and 8.2. The enzyme activity increased linearly between 30 and 44°C. It was stable for several months at −20°C. Magnesium was essential for activity, but the rate attainable with Mn²⁺ was at least as great as that due to Mg²⁺. No other divalent cation was able to substitute for Mg²⁺ or Mn²⁺. Neither low nor high Ca²⁺ concentrations significantly affected the enzymatic activity. Substrate specificity studies showed that ATP was the preferred substrate followed by CTP (46% of the rate produced by ATP). Hydrolysis of GTP, UTP, ITP and ADP was less than 10% of the rate seen with ATP. No phosphatase, pyrophosphatase, phosphodiesterase, hexokinase, phosphofructokinase or adenylate cyclase activity could be detected in this enzyme preparation. Calmodulin, which stimulates the (Ca²⁺ + Mg²⁺)-ATPase of the human erythrocyte membrane, failed to enhance the Mg²⁺-ATPase activity. Of considerable interest, the activity of this Mg²⁺-ATPase was enhanced approximately 5-fold by low concentrations of mercuric ion, p-hydroxymercuribenzoate and DTNB, but was much less sensitive to iodoacetamide.     

Partial purification and characterization of a novel Mg2+- dependent ATPase present in the cytosol from human erthrocytes

Mg²⁺-ATPase activity was identified in the cytosol of human erythrocytes. A partial purification of this activity was achieved by an initial DEAE-Sephadex column chromatography, followed by gel filtration on Sephadex G-100 and then a second DEAE-Sephadex chromatography procedure. The enzyme appeared in the void volume of the Sephadex G-100 column and was retained on an Amicon XM100A ultrafiltration membrane. The molecular weight of the enzyme was estimated to be 113 000 from SDS gels. The above purification protocol yielded an enzyme with an optimal pH between 7.6 and 8.2. The enzyme activity increased linearly between 30 and 44°C. It was stable for several months at ?20°C. Magnesium was essential for activity, but the rate attainable with Mn²⁺ was at least as great as that due to Mg²⁺. No other divalent cation was able to substitute for Mg²⁺ or Mn²⁺. Neither low nor high Ca²⁺ concentrations significantly affected the enzymatic activity. Substrate specificity studies showed that ATP was the preferred substrate followed by CTP (46% of the rate produced by ATP). Hydrolysis of GTP, UTP, ITP and ADP was less than 10% of the rate seen with ATP. No phosphatase, pyrophosphatase, phosphodiesterase, hexokinase, phosphofructokinase or adenylate cyclase activity could be detected in this enzyme preparation. Calmodulin, which stimulates the (Ca²⁺ + Mg²⁺)-ATPase of the human erythrocyte membrane, failed to enhance the Mg²⁺-ATPase activity. Of considerable interest, the activity of this Mg²⁺-ATPase was enhanced approximately 5-fold by low concentrations of mercuric ion, p-hydroxymercuribenzoate and DTNB, but was much less sensitive to iodoacetamide.     

Properties of 1-phosphofructokinase from Pseudomonas putida.

The 1-phosphofructokinase (1-PFK, EC 2.7.1.56) from Pseudomonas putida was partially purified by a combination of (NH4)2SO4 fractionation and DEAE-Sephadex column chromatography. In its kinetic properties, this enzyme resembled the 1-PFK's from other bacteria. With the substrates fructose-1-phosphate (F-1-P) and adenosine triphosphate (ATP) Michaelis-Menten kinetics were observed, the Km for one substrate being unaffected by a variation in the concentration of the other substrate. At pH 8.0, the Km values for F-1-P and ATP were 1.64 X 10(-4) M and 4.08 X 10(-4) M, respectively. At fixed concentrations of F-1-P and ATP, an increase in the Mg2+ resulted in sigmoidal kinetics. Activity was inhibited by ATP when the ratio of ATP:Mg2+ was greater than 0.5 suggesting that ATP:2 Mg2+ was the substrate and free ATP was inhibitory. Activity of 1-PFK was stimulated by K+ and to a lesser extent by NH4+ and Na+. The reaction rate was unaffected by 2 mM K2HPO4, pyruvate, phosphoenolpyruvate, adenosine monophosphate, adenosine 3',5'-cyclic monophosphate, fructose-6-phosphate, glucose-6-phosphate, 6-phosphogluconate, 2-keto-3-deoxy-6-phosphogluconate, or citrate. The results indicated that the 1-PFK from P. putida was not allosterically regulated by a number of metabolites which may play an important role in the catabolism of D-fructose.     

Biochemical Studies on “Bakanae” Fungus. Part 54

Phosphate compounds in potato tubers were fractionated by ion-exchange chromatography. By the use of radio-phosphorus, the following compounds were tentatively identified to be present in potato tubers: G-1-P, G-6-P, F-6-P, FDP, PGA, AMP, UMP, ADP, UDPX, UDPG and ATP. When the tubers were stored at a low temperature, the contents of organic phosphate compounds showed a considerable increase over those of potatoes stored at a high temperature. However, the relative composition of each component was not much influenced by the storage temperature except ATP which showed a great increase at a lower temperature.