Temperature and the regulation of enzyme activity in poikilotherms. Properties of lungfish fructose diphosphatase

1. The properties of fructose diphosphatase from liver of South American lungfish (Lepidosiren paradoxa) were examined. 2. Saturation curves for substrate (fructose diphosphate) and both cofactors (Mn(2+) and Mg(2+)) are sigmoidal and Hill plots of these results suggest about 2 interacting substrate and cofactor sites/molecule of enzyme. 3. Mn(2+) is an efficient positive modulator of the enzyme and K(a) for Mn(2+) is about 20-30-fold lower than the K(a) for Mg(2+). 4. Lungfish fructose diphosphatase is inhibited by low concentrations of AMP, and the affinity of the enzyme for AMP is insensitive to temperature. 5. The affinities of fructose diphosphatase for fructose diphosphate and Mn(2+) appear to be dependent on temperature, whereas affinity for Mg(2+) is temperature-independent. 6. The pH optimum of the enzyme depends on the presence of the particular cofactor. As pH increases, the K(a) values of both cations are lowered, maximum velocities are increased and the saturation curves for cofactor become hyperbolic. 7. The possible roles of these ions, pH and substrate in the modulation of fructose diphosphatase and gluconeogenic activity in the lungfish are discussed in relation to aestivation and temperature adaptation.     

The inhibition of skeletal-muscle fructose 1,6-diphosphatase by adenosine monophosphate

1. The present study extends the finding of Krebs & Woodford (1965) that muscle fructose diphosphatase is more sensitive to AMP inhibition than liver fructose diphosphatase. 2. Hen breast fructose diphosphatase has a K(i) for AMP of 0.1mum; the plot of percentage inhibition is non-sigmoid and the reciprocal plot of activity against AMP concentration is sometimes linear. 3. Percentage inhibition plots for other muscle fructose diphosphatases are sigmoid curves which exhibit different threshold responses to the AMP concentration. 4. The intracellular content of AMP in all muscles tested exceeds the inhibition concentration range of AMP. 5. The sensitivity of muscle fructose diphosphatase to AMP inhibition is decreased by the presence of Mg(2+) or Mn(2+) ions; in the presence of Mn(2+) the inhibition curve for hen breast fructose diphosphatase becomes sigmoid. 6. From the formation constants for the Mg(2+) and Mn(2+) chelates, the effect of these ions in chelation of AMP can be calculated. Although chelation of AMP can explain the Mg(2+) effect, it cannot explain the marked relief of AMP inhibition by Mn(2+). 7. It is suggested that Mn(2+) has a specific effect on this enzyme which reduces the sensitivity to AMP inhibition.     

The functional significance of variants of fructose 1,6-diphosphatase in the gill and hypodermis of a marine crustacean. A kinetic study

1. In the hypodermis and gill of the Crustacea fructose 1,6-diphosphatase (EC 3.1.3.11) functions at a primary branch point between glycogen and chitin synthesis. In these tissues of the Arctic king-crab, Paralithodes camtchatica, fructose diphosphatase occurs in two electrophoretically distinguishable forms. 2. Fructose diphosphatase I (pI7.2-7.5) accounts for 70 and 10% of total fructose diphosphatase activity in the hypodermis and gill respectively, whereas fructose diphosphatase II (pI5.3) accounts for 30 and 90% of the total activity in the two tissues. Both forms display a neutral pH optimum, have an absolute requirement for a bivalent cation, and are potently inhibited by high concentrations of AMP and substrate. 3. Fructose 1,6-diphosphate saturation follows Michaelis-Menten kinetics for both fructose diphosphatases; the K(m) (fructose diphosphate) for fructose diphosphatase I is somewhat higher than for fructose diphosphatase II. In the presence of 50-200mm-K(+), the K(m) (fructose diphosphate) increases and at high concentrations of K(+) fructose diphosphate saturation follows sigmoidal kinetics. 4. UDP-N-acetylglucosamine and UDP-glucose at high concentrations specifically and potently inhibit fructose diphosphatase II, but do not significantly affect fructose diphosphatase I activity. 5. Low concentrations of UDP-N-acetylglucosamine activate fructose diphosphatase II by a decrease in the apparent K(m) (fructose diphosphate), but fructose diphosphatase I is again refractory to UDP-N-acetylglucosamine under these conditions. 6. In the presence of K(+) and UDP-N-acetylglucosamine, fructose diphosphatase II is able to compete for limiting fructose diphosphate about three times more effectively than is fructose diphosphatase I. 7. AMP inhibition of both forms of the enzyme is subject to three independent variables: (a) alkaline pH increases the K(i) (AMP), (b) K(+) decreases the K(i), increases the sigmoidicity of inhibition kinetics, increases the maximum inhibition attained, and abolishes the effect of pH on AMP inhibition, and (c) Mg(2+) strongly de-inhibits AMP-inhibited fructose diphosphatase. 8. It is postulated that the presence of two forms of fructose diphosphatase aids controlled channelling of carbon through the fructose diphosphatase ;bottleneck' either towards glycogen synthesis or chitin synthesis, but not towards both simultaneously.     

Some aspects of the kinetics of rat liver pyruvate carboxylase

1. The kinetics of rat liver pyruvate carboxylase were examined and the effect of various agents as activators or inhibitors determined. 2. Essentially similar results were obtained in comparisons of kinetics determined by a radioactivity method involving extracts of acetone-dried powders from whole livers and with a spectrophotometric assay using partially purified enzyme from the mitochondrial fraction. Activity per g of liver from fed or starved rats assayed under optimum substrate and activator conditions was 3 or 6 mumol of oxaloacetate formed/min at 30 degrees C, respectively. 3. The enzyme exhibited cold-lability and lost activity on standing, even in 1.5m-sucrose. 4. The K(m) towards pyruvate was about 0.33mm and towards bicarbonate 4.2mm. K(m) towards MgATP(2-) was 0.14mm. Mg(2+) ions activated the enzyme, in addition to their role in MgATP(2-) formation. From calculations of likely concentrations of free Mg(2+) in the assay medium a K(a) towards Mg(2+) of about 0.25mm was deduced. Mn(2+) also activated the enzyme as well as Mg(2+), but at much lower concentrations. It appeared to be inhibitory when concentrations of free Mn(2+) as low as 0.1mm were present. 5. Excess of ATP is inhibitory, and this appears at least in part independent of the trapping of Mg(2+). 6. Both Co(2+) and Zn(2+) were inhibitory; 2mol of the latter appeared to be bound even in the presence of excess of Mg(2+) and the inhibition was time-dependent. 7. Ca(2+) inhibited by competition with Mg(2+) (K(i) about 0.38mm). The inhibition due to Ca(2+) was less pronounced when activation was with Mn(2+). Inhibition by Ca(2+) and ATP appeared to be additive. 8. Hill plots suggested that no interactions occurred between ATP-binding sites. Although similar plots for total Mg(2+) gave n=3.6, no conclusions could be drawn due to the chelation of the cation with other components of the assay. Similar difficulties arose in assessing the values for Ca(2+). 9. The enzyme was inactive in the absence of acetyl-CoA and showed a sigmoidal response in its presence. K(a) was about 0.1mm with possibly up to four binding sites. Malonyl-CoA was a competitive inhibitor, with K(i) 0.01mm. 10. There was no apparent inhibition by glucose, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-diphosphate, acetoacetate, beta-hydroxybutyrate, malate, aspartate, pyruvate, palmitoylcarnitine, octanoate, glutathione, butacaine, triethyltin or potassium chloride under the conditions used. Inhibition was found with citrate (possibly by chelation) and adenosine, and also by phosphoenolpyruvate, AMP, ADP and cyclic AMP, K(i) towards the last four being 0.55, 0.76, 0.25 and 1.4mm respectively.     

Temperature and the regulation of enzyme activity in poikilotherms. Regulatory properties of fructose diphosphatase from muscle of the Alaskan king-crab

1. The properties of fructose diphosphatase from skeletal muscle of the Alaskan king-crab (Paralithodes camtschatica) were examined over the physiological temperature range of the animal. 2. King-crab muscle fructose diphosphatase is first activated by Na(+) and NH(4) (+) and is then partially inhibited by these cations at concentrations higher than 10mm at 0 degrees , 8 degrees and 15 degrees C. Enzyme activity is stimulated by K(+) at 0 degrees C, but is curtailed at 8 degrees C and 15 degrees C, an effect that could render rate independent of temperature. 3. Affinity for substrate increases with decreasing temperature; below the temperature of acclimatization, K(m) for fructose 1,6-diphosphate increases, resulting in a complex U-shaped temperature-K(m) curve. 4. King-crab muscle fructose diphosphatase is inhibited by low concentrations of AMP. As with enzymes of other poikilotherms, inhibition by AMP is sensitive to temperature; the enzyme is least sensitive to inhibition by AMP near the temperature of acclimatization. 5. The affinity of fructose diphosphatase for fructose 1,6-diphosphate is enhanced by phosphoenolpyruvate, and this activation is temperature-sensitive; 0.5mm-phosphoenolpyruvate causes a sevenfold decrease in K(m) for fructose 1,6-diphosphate at 15 degrees C but a 25-fold decrease at 0 degrees C. 6. Phosphoenolpyruvate appears to decrease the affinity of king-crab muscle fructose diphosphatase for AMP at low temperature, whereas at the higher temperature it appears to enhance inhibition by AMP. Phosphoenolpyruvate was not observed to cause a reversal of inhibition of fructose diphosphatase activity by AMP. The identification of phosphoenolpyruvate as an activator of a rate-limiting step in gluconeogenesis permits the suggestion of a coupling of the controlling mechanisms of several steps in the glycolytic and gluconeogenic chains. 7. These findings suggest mechanisms for the maintenance and regulation of control of fructose diphosphatase activity in king-crab skeletal muscle at low temperature and under conditions that favour concomitant activity of phosphofructokinase.     

Kinetic properties of rat liver pyruvate kinase at cellular concentrations of enzyme, substrates and modifiers

Kinetic properties of rat liver pyruvate kinase type I at pH7.5 and 6.5 were studied with physiological ranges of substrates, modifiers and Mg(2+) concentrations at increasing enzyme concentrations, including the estimated cellular concentrations (approx. 0.1mg/ml). Enzyme properties appear unaffected by increased enzyme concentration if phosphoenolpyruvate, fructose 1,6-diphosphate and inhibitors are incubated with enzyme before starting the reaction with ADP. Our data suggest that minimum cellular concentrations of MgATP and l-alanine provide virtually complete inhibition of pyruvate kinase I at pH7.5. The most likely cellular control of existing pyruvate kinase I results from the strong restoration of enzyme activity by the small physiological amounts of fructose 1,6-diphosphate. Decreasing the pH to 6.5 also restores pyruvate kinase activity, but to only about one-third of its activity in the presence of fructose 1,6-diphosphate. Neither pyruvate nor 2-phosphoglycerate at cellular concentrations inhibit the enzyme significantly.     

Kinetic studies on the regulation of rabbit liver pyruvate kinase

Two kinetically distinct forms of pyruvate kinase (EC 2.7.1.40) were isolated from rabbit liver by using differential ammonium sulphate fractionation. The L or liver form, which is allosterically activated by fructose 1,6-diphosphate, was partially purified by DEAE-cellulose chromatography to give a maximum specific activity of 20 units/mg. The L form was allosterically activated by K(+) and optimum activity was recorded with 30mm-K(+), 4mm-MgADP(-), with a MgADP(-)/ADP(2-) ratio of 50:1, but inhibition occurred with K(+) concentrations in excess of 60mm. No inhibition occurred with either ATP or GTP when excess of Mg(2+) was added to counteract chelation by these ligands. Alanine (2.5mm) caused 50% inhibition at low concentrations of phosphoenolpyruvate (0.15mm). The homotropic effector, phosphoenolpyruvate, exhibited a complex allosteric pattern (n(H)=2.5), and negative co-operative interactions were observed in the presence of low concentrations of this substrate. The degree of this co-operative interaction was pH-dependent, with the Hill coefficient increasing from 1.1 to 3.2 as the pH was raised from 6.5 to 8.0. Fructose 1,6-diphosphate interfered with the activation by univalent ions, markedly decreased the apparent K(m) for phosphoenolpyruvate from 1.2mm to 0.2mm, and transformed the phosphoenolpyruvate saturation curve into a hyperbola. Concentrations of fructose 1,6-diphosphate in excess of 0.5mm inhibited this stimulated reaction. The M or muscle-type form of the enzyme was not activated by fructose 1,6-diphosphate and gave a maximum specific activity of 0.3 unit/mg. A Michaelis-Menten response was obtained when phosphoenolpyruvate was the variable substrate (K(m)=0.125mm), and this form was inhibited by ATP, as well as alanine, even in the presence of excess of Mg(2+).     

The activities of fructose diphosphatase in flight muscles from the bumble-bee and role of this enzyme in heat generation

1. The maximum catalytic activities of fructose diphosphatase from flight muscles of bumble-bees (Bombus spp.) are at least 30-fold those reported for the enzyme from other tissues. The maximum activity of fructose diphosphatase in the flight muscle of any particular bee is similar to that of phosphofructokinase in the same muscle, and the activity of hexokinase is similar to or greater than the activity of phosphofructokinase. There is no detectable activity of glucose 6-phosphatase and only a very low activity of glucose 6-phosphate dehydrogenase in these muscles. The activities of both fructose diphosphatase and phosphofructokinase vary inversely with the body weight of the bee, whereas that of hexokinase is relatively constant. 2. There is no significant hydrolysis of fructose 1-phosphate, fructose 6-phosphate, glucose 1,6-diphosphate and glycerol 3-phosphate by extracts of bumble-bee flight muscle. 3. Fructose 1,6-diphosphatase from bumble-bee flight muscle and from other muscles is inhibited by Mn(2+) and univalent cations; the potency of inhibition by the latter varies in the order Li(+)>Na(+)>K(+). However, the fructose diphosphatase from bumble-bee flight muscle is different from the enzyme from other tissues in that it is not inhibited by AMP. 4. The contents of ATP, hexose monophosphates, fructose diphosphate and triose phosphates in bumble-bee flight muscle showed no significant changes between rest and flight. 5. It is proposed that both fructose diphosphatase and phosphofructokinase are simultaneously active and catalyse a cycle between fructose 6-phosphate and fructose diphosphate in resting bumble-bee flight muscle. Such a cycle would produce continuous hydrolysis of ATP, with the release of energy as heat, which would help to maintain the thoracic temperature during rest periods at a level adequate for flight.     

The effect of monovalent and divalent cations on the activity of Streptococcus lactis C10 pyruvate kinase.

The pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis C10 had an obligatory requirement for both a monovalent cation and divalent cation. NH+4 and K+ activated the enzyme in a sigmoidal manner (nH =1.55) at similar concentrations, whereas Na+ and Li+ could only weakly activate the enzyme. Of eight divalent cations studied, only three (Co2+, Mg2+ and Mn2+) activated the enzyme. The remaining five divalent cations (Cu2+, Zn2+, Ca2+, Ni2+ and Ba2+) inhibited the Mg2+ activated enzyme to varying degrees. (Cu2+ completely inhibited activity at 0.1 mM while Ba2+, the least potent inhibitor, caused 50% inhibition at 3.2 mM). In the presence of 1 mM fructose 1,6-diphosphate (Fru-1,6-P2) the enzyme showed a different kinetic response to each of the three activating divalent cations. For Co2+, Mn2+ and Mg2+ the Hill interaction coefficients (nH) were 1.6, 1.7 and 2.3 respectively and the respective divalent cation concentrations required for 50% maximum activity were 0.9, 0.46 and 0.9 mM. Only with Mn2+ as the divalent cation was there significatn activity in the absence of Fru-1,6-P2. When Mn2+ replaced Mg2+, the Fru-1,6-P2 activation changed from sigmoidal (nH = 2.0) to hyperbolic (nH = 1.0) kinetics and the Fru-1,6-P2 concentration required for 50% maximum activity decreased from 0.35 to 0.015 mM. The cooperativity of phosphoenolpyruvate binding increased (nH 1.2 to 1.8) and the value of the phosphoenolpyruvate concentration giving half maximal velocity decreased (0.18 to 0.015 mM phosphoenolyruvate) when Mg2+ was replaced by Mn2+ in the presence of 1 mM Fru-1,6-P2. The kinetic response to ADP was not altered significantly when Mn2+ was substituted for Mg2+. The effects of pH on the binding of phosphoenolpyruvate and Fru-1,6-P2 were different depending on whether Mg2+ or Mn2+ was the divalent cation.     

The purification and properties of pig spleen phosphofructokinase.

Pig spleen phosphofructokinase has been purified 800-fold with a yield of 17%. Two isoenzymes that appear to be kinetically identical can be separated by DEAE-cellulose column chromatography. In common with the enzyme from other mammalian sources, the spleen enzyme has a pH optimum of 8.2. At pH 7.0 it displays sigmoidal kinetics with respect to fructose 6-phosphate concentration but its co-operative behaviour is very dependent on pH, protein concentration and the concentration of MgATP. MgGTP and MgITP can replace MgATP as phosphate donors but, unlike MgATP, these nucleotides do not cause significant inhibition. Mn2+ and Co2+ (as the metal ion-ATP complexes) act as cofactors and in the free form are far more inhibitory than free Mg2+. The spleen enzyme responds to a wide variety of potential effector molecules: ADP, AMP, cyclic AMP, aspartate, NH4+, fructose 6-phosphate, fructose 1,6-diphosphate and Pi all act as either activators or protectors, whereas Mg-ATP, Mg2+, citrate, phosphoenol-pyruvate and the phosphoglucerates are inhibitors.     

Adipose-tissue pyruvate kinase. Properties and interconversion of two active forms

1. Extraction of rat epididymal adipose tissue with buffer containing EDTA yields a pyruvate kinase, provisionally called PyK-A, the properties of which resemble in several respects those of the allosteric pyruvate kinase of liver. These properties include co-operative interactions with phosphoenolpyruvate, Mg(2+), K(+), NH(4) (+) and ATP, and sensitivity to activation by fructose 1,6-diphosphate. 2. Extraction in the absence of EDTA yields predominantly a form, PyK-B, that shows both normal Michaelis-Menten kinetics with phosphoenolpyruvate, Mg(2+) and ATP, and co-operative interactions with K(+) and NH(4) (+); this form is insensitive towards fructose 1,6-diphosphate. 3. Both forms yield simple kinetics with ADP, though K(m) values differ in the two systems. In all cases where co-operativity has been demonstrated, Hill-plot n values are between 1.4 and 2.0. 4. The conversion of PyK-A into PyK-B is mediated specifically by fructose 1,6-diphosphate; the reverse reaction is occasioned by EDTA, ATP or citrate. It is thought that a bivalent cation may be involved in this interconversion. 5. Attempts at partial purification have revealed that the enzyme resembles the pyruvate kinase of skeletal muscle, rather than that of liver, in its solubility in ammonium sulphate and elution from DEAE-cellulose. 6. The relevance of these properties in the regulation of pyruvate kinase activity in vivo in adipose tissue is discussed.     

Thermodynamic linked-function analysis of Mg(2+)-activated yeast pyruvate kinase.

Yeast pyruvate kinase (YPK) is regulated by intermediates of the glycolytic pathway [e.g., phosphoenolpyruvate (PEP), fructose 1,6-bisphosphate (FBP), and citrate] and by the ATP charge of the cell. Recent kinetic and thermodynamic data with Mn(2+)-activated YPK show that Mn(2+) mediates the allosteric communication between the substrate, PEP, and the allosteric effector, FBP [Mesecar, A., and Nowak, T. (1997) Biochemistry 36, 6792, 6803]. These results indicate that divalent cations modulate multiligand interactions, and hence cooperativity with YPK. The nature of multiligand interactions on YPK was investigated in the presence of the physiological divalent activator Mg(2+). The binding interactions of PEP, Mg(2+), and FBP were monitored by fluorescence spectroscopy. The binding data were subject to thermodynamic linked-function analysis to determine the magnitudes of the multiligand interactions governing the allosteric activation of YPK. The two ligand coupling free energies between PEP and Mg(2+), PEP and FBP, and FBP and Mg(2+) are 0.88, -0.38, and -0.75 kcal/mol, respectively. The two-ligand coupling free energies between PEP and Mn(2+) and FBP and Mn(2+) are more negative than those with Mg(2+) as the cation. This indicates that the interactions between the divalent cation and PEP with YPK are different for Mg(2+) and Mn(2+) and that the interaction is not simply electrostatic in nature, as originally hypothesized. The magnitude of the heterotropic interaction between the metal and FBP is similar with Mg(2+) and Mn(2+). The simultaneous binding of Mg(2+), PEP, and FBP to YPK is favored by 3.21 kcal/mol compared to independent binding. This complex is destabilized by 3.30 kcal/mol relative to the analogous YPK-Mn(2+)-PEP-FDP complex. Interpretation of K(d) values when cooperative binding occurs must be done with care as these are not simple thermodynamic constants. These data demonstrate that the divalent metal, which activates phosphoryl transfer in YPK, plays a key role in modulating the various multiligand interactions that define the overall allosteric properties of the enzyme.     

Origin of cooperativity in the activation of fructose-1,6-bisphosphatase by Mg2+

Fructose-1,6-bisphosphatase requires a divalent metal cation for catalysis, Mg(2+) being its most studied activator. Phosphatase activity increases sigmoidally with the concentration of Mg(2+), but the mechanistic basis for such cooperativity is unknown. Bound magnesium cations can interact within a single subunit or between different subunits of the enzyme tetramer. Mutations of Asp(118), Asp(121), or Glu(97) to alanine inactivate the recombinant porcine enzyme. These residues bind directly to magnesium cations at the active site. Three different hybrid tetramers of fructose-1,6-bisphosphatase, composed of one wild-type subunit and three subunits bearing one of the mutations above, exhibit kinetic parameters (K(m) for fructose-1,6-bisphosphate, 1.1-1.8 microm; K(a) for Mg(2+), 0.34-0.76 mm; K(i) for fructose-2,6-bisphosphate, 0.11-0.61 microm; and IC(50) for AMP, 3.8-7.4 microm) nearly identical to those of the wild-type enzyme. Notwithstanding these similarities, the k(cat) parameter for each hybrid tetramer is approximately one-fourth of that for the wild-type enzyme. Evidently, each subunit in the wild-type tetramer can independently achieve maximum velocity when activated by Mg(2+). Moreover, the activities of the three hybrid tetramers vary sigmoidally with the concentration of Mg(2+) (Hill coefficients of approximately 2). The findings above are fully consistent with a mechanism of cooperativity that arises from within a single subunit of fructose-1,6-bisphosphatase.     

The requirement for bivalent cations in formation of nicotinamide–adenine dinucleotide by nicotinamide mononucleotide adenylyltransferase of pig-liver nuclei

1. The requirement for bivalent cations in catalysis of NAD formation from ATP and NMN in the presence of NMN adenylyltransferase of pig-liver nuclei was studied. Rates of NAD formation in the presence of the activating cations Cd(2+), Mn(2+), Mg(2+), Zn(2+), Co(2+) and Ni(2+) were approximately a linear function of heats of hydration of the corresponding ions. Ba(2+), Sr(2+), Ca(2+), Cu(2+) and Be(2+) did not activate the enzyme; Be(2+) inhibited the reaction in the presence of Mg(2+) and, to a greater extent, in the presence of Ni(2+). 2. Michaelis constants for NAD formation, measured in a coupled assay with NMN adenylyltransferase and alcohol dehydrogenase at pH8.0 and 25 degrees , in the presence of 3mm concentrations of the unvaried reactants, were 88+/-7mum-ATP, 42+/-4mum-NMN and 85+/-4mum-Mg(2+). The results at this pH and at pH7.5 were consistent with mechanisms in which Mg(2+)-ATP complex is a reactant and free ATP a competitive inhibitor. 3. Formation of nicotinamide-hypoxanthine dinucleotide from NMN and ITP in the presence of the transferase was also more rapid with Ni(2+) and Co(2+) than with Mg(2+).     

Fructokinase (Fraction IV) of Pea Seeds

A fructokinase (EC 2.7.1.4) was obtained from pea (Pisum sativum L.) seeds. This enzyme, termed fructokinase (fraction IV), was specific for fructose as substrate and had little activity with glucose or mannose. Excess fructose inhibited the enzyme at the optimum pH (8.2) but not at pH 6.6. MgATP was inhibitory at pH 6.6. The apparent Michaelis-Menten constants at pH 8.2 were 0.057 mm for fructose and 0.10 mm for MgATP. Mg(2+) ions were essential for activity; Mn(2+) could partially replace Mg(2+). Fructokinase (fraction IV) had a requirement for K(+) ions which could be substantially replaced by Rb(+) or NH(4) (+) but not by Na(+). The enzyme was inhibited by MgADP. The possible significance of fructokinase (fraction IV) in plant carbohydrate metabolism is discussed.     

A specific sucrose phosphatase from plant tissues

1. A phosphatase that hydrolyses sucrose phosphate (phosphorylated at the 6-position of fructose) was isolated from sugar-cane stem and carrot roots. With partially purified preparations fructose 6-phosphate, glucose 6-phosphate, fructose 1-phosphate, glucose 1-phosphate and fructose 1,6-diphosphate are hydrolysed at between 0 and 2% of the rate for sucrose phosphate. 2. The activity of the enzyme is increased fourfold by the addition of Mg(2+) ions and inhibited by EDTA, fluoride, inorganic phosphate, pyrophosphate, Ca(2+) and Mn(2+) ions. Sucrose (50mm) reduces activity by 60%. 3. The enzyme exhibits maximum activity between pH6.4 and 6.7. The Michaelis constant for sucrose phosphate is between 0.13 and 0.17mm. 4. At least some of the specific phosphatase is associated with particles having the sedimentation properties of mitochondria. 5. A similar phosphatase appears to be present in several other plant species.     

A comparison of the properties of the pyruvate kinases of the fat body and flight muscle of the adult male desert locust

1. The pyruvate kinases of the desert locust fat body and flight muscle were partially purified by ammonium sulphate fractionation. 2. The fat-body enzyme is allosterically activated by very low (1mum) concentrations of fructose 1,6-diphosphate, whereas the flight-muscle enzyme is unaffected by this metabolite at physiological pH. 3. Flight-muscle pyruvate kinase is activated by preincubation at 25 degrees for 5min., whereas the fat-body enzyme is unaffected by such treatment. 4. Both enzymes require 1-2mm-ADP for maximal activity and are inhibited at higher concentrations. With the fat-body enzyme inhibition by ADP is prevented by the presence of fructose 1,6-diphosphate. 5. Both enzymes are inhibited by ATP, half-maximal inhibition occurring at about 5mm-ATP. With the fat-body enzyme ATP inhibition can be reversed by fructose 1,6-diphosphate. 6. The fat-body enzyme exhibits maximal activity at about pH7.2 and the activity decreases rapidly above this pH. This inactivation at high pH is not observed in the presence of fructose 1,6-diphosphate, i.e. maximum stimulating effects of fructose 1,6-diphosphate are observed at high pH. The flight-muscle enzyme exhibits two optima, one at about pH7.2 as with the fat-body enzyme and the other at about pH8.5. Stimulation of the enzyme activity by fructose 1,6-diphosphate was observed at pH8.5 and above.     

Purification and properties of spinach leaf cytoplasmic fructose-1,6-bisphosphatase.

Cytoplasmic fructose-1,6-bisphosphatase has been purified from spinach leaves to apparent homogeneity. The enzyme is a tetramer of molecular weight about 130,000. At pH 7.5, the Km for fructose 1.6-bisphosphate was 2.5 micron, and for MgCl2 0.13 mM; the enzyme was specific for fructose 1,6-bisphosphate. Saturation with Mg2+ was achieved with lower concentrations at pH 8 than at pH 7. AMP and high concentrations of fructose 1,6-bisphosphate inhibited enzyme activity. Ammonium sulfate relieved the latter inhibition but was itself inhibitory when substrate concentrations were low. Acetylation studies demonstrated that the AMP regulatory site was distinct from the catalytic site. Cytoplasmic fructose-1,6-bisphosphatase may contribute to the regulation of sucrose biosynthesis in plant leaves.     

Activation of DNA-hydrolyzing antibodies from the sera of autoimmune-prone MRL-lpr/lpr mice by different metal ions

We have shown previously that electrophoretically and immunologically homogeneous polyclonal IgGs from the sera of autoimmune-prone MRL mice possess DNase activity. Here we have analyzed for the first time activation of DNase antibodies (Abs) by different metal ions. Polyclonal DNase IgGs were not active in the presence of EDTA or after Abs dialysis against EDTA, but could be activated by several externally added metal (Me(2+)) ions, with the level of activity decreasing in the order Mn(2+)> or =Mg(2+)>Ca(2+)> or =Cu(2+)>Co(2+)> or =Ni(2+)> or =Zn(2+), whereas Fe(2+) did not stimulate hydrolysis of supercoiled plasmid DNA (scDNA) by the Abs. The dependencies of the initial rate on the concentration of different Me(2+) ions were generally bell-shaped, demonstrating one to four maxima at different concentrations of Me(2+) ions in the 0.1-12 mM range, depending on the particular metal ion. In the presence of all Me(2+) ions, IgGs pre-dialyzed against EDTA produced only the relaxed form of scDNA and then sequence-independent hydrolysis of relaxed DNA followed. Addition of Cu(2+), Zn(2+), or Ca(2+) inhibited the Mg(2+)-dependent hydrolysis of scDNA, while Ni(2+), Co(2+), and Mn(2+) activated this reaction. The Mn(2+)-dependent hydrolysis of scDNA was activated by Ca(2+), Ni(2+), Co(2+), and Mg(2+) ions but was inhibited by Cu(2+) and Zn(2+). After addition of the second metal ion, only in the case of Mg(2+) and Ca(2+) or Mn(2+) ions an accumulation of linear DNA (single strand breaks closely spaced in the opposite strands of DNA) was observed. Affinity chromatography on DNA-cellulose separated DNase IgGs into many subfractions with various affinities to DNA and very different levels of the relative activity (0-100%) in the presence of Mn(2+), Ca(2+), and Mg(2+) ions. In contrast to all human DNases having a single pH optimum, mouse DNase IgGs demonstrated several pronounced pH optima between 4.5 and 9.5 and these dependencies were different in the presence of Mn(2+), Ca(2+), and Mg(2+) ions. These findings demonstrate a diversity of the ability of IgG to function at different pH and to be activated by different optimal metal cofactors. Possible reasons for the diversity of polyclonal mouse abzymes are discussed.