Pyruvate kinase variants of the Alaskan king-crab. Evidence for a temperature-dependent interconversion between two forms having distinct and adaptive kinetic properties

1. Pyruvate kinase of Alaskan king-crab leg muscle exists in two kinetically distinct forms, each of which displays a different temperature-dependence in the K_m for phosphoenolpyruvate. 2. A `cold' variant of the enzyme has hyperbolic kinetics and exhibits a minimal K<sub>m</sub> for substrate at 5°. At physiological concentrations of phosphoenolpyruvate the `cold' enzyme is active only below 10°. A `warm' pyruvate kinase has a minimal K<sub>m</sub> for substrate at about 12°. This enzyme displays sigmoidal kinetics and is likely to be inactive, at physiological substrate concentrations, at temperatures below 9°. 3. The combined activities of these two pyruvate kinases yield highly temperature-independent rates of catalysis, at physiological substrate concentrations, over the range of habitat temperatures encountered by the organism, namely 4–12°. 4. The two variants of pyruvate kinase do not appear to be isoenzymes in the conventional sense. Electrophoretic and electrofocus analyses revealed only single peaks of activity. 5. The results suggest that the `warm' pyruvate kinase and the `cold' pyruvate kinase are formed by a temperature-dependent interconversion of one protein species. This interconversion has major adaptive significance: as the temperature is lowered the `warm' enzyme is converted into the `cold' enzyme; the opposite situation obtains when the temperature is raised. Temperature changes thus mimic the effects noted for fructose 1,6-diphosphate on certain mammalian pyruvate kinases.     

Purification and properties of pyruvate kinase type M2 from rat lung

(1) Pyruvate kinase type M₂ from rat lung has been purified 840-fold with an overall yield of 20%. The enzyme gave a single band upon SDS-electrophoresis and isoelectrofocusing and had a specific activity of 1340 U/mg protein. The homotetramer of M_r = 224 000 and an isoelectric point of pH 5.8 had an amino acid composition closely resembling that of other pyruvate kinase isoenzymes type M₂, excepts that of the chicken liver. The enzyme was crystallized. (2) The enzyme has its pH optimum at pH 6.5. The K_0.5 value for phosphoenolpyruvate is 0.26 mM (n_H = 1.81) which decreases in the presence of 0.2 mM fructose 1,6-bisphosphate to 0.056 mM (n_H = 1.06). 1 μM fructose 1,6-bisphosphate activates the enzyme at 0.1 mM phosphoenolpyruvate half-maximally. The K_m value for ADP at 1 mM phosphoenolpyruvate is 0.4 mM. The K_m value for other nucleoside diphosphates increases in the order ADP<GDP<IDP<UDP. (3) No evidence for an interconversion of pyruvate kinase type M₂ from rat or chicken lung was found. The enzyme was neither a substrate for the cAMP-dependent protein kinase from rabbit muscle nor for the cAMP-independent protein kinase from chicken liver. Since pyruvate kinase type M₂ from chicken liver is inactivated by phosphorylation catalyzed by a cAMP-independent protein kinase (Eigenbrodt, E., Abdel-Fattah Mostafa, M. and Schoner, W. (1977) Hoppe-Seyler's Z. Physiol. Chem. 358, 1047–1055) we suggest that the interconvertible form of pyruvate kinase type M₂ may represent a separate form of the pyruvate kinase type M₂ family.     

Ribonucleoprotein complexes of pyruvate kinase from human skeletal muscle

Summary About 25% of total pyruvate kinase activity in muscle appears in a bound form which is insoluble in water or diluted salt solutions at pH 5.8. That activity is associated with the ribonuc-leoprotein complexes and is soluble at high ionic strength. A procedure is described for the purification and crystallization of this enzyme form herein called pyruvate kinase M_B and water soluble form M_A.Crystalline nucleoproteins are composed of active and inactive RNA-protein complexes with varying RNA content. By fractional crystallization and gel filtration a number of crystalline complexes were separated, two of them highly purified. One preparation was homogenous, contained 0.5% RNA and had a specific activity of 265 U/mg protein, the other one 10% RNA and 200 U/mg protein respectively.Forms M_A and M_B share the same protein as shown in immunodiffusion test with the anti-M_A sera. They differ in solubility and stability in diluted solutions. In Tris-HCl buffer, pH 7.6 form M_B is rapidly inactivated whereas form M_A is quite stable under the same conditions. Both forms have different K_m for phosphoenolpyruvate and ADP and V_max as well.Digestion of pyruvate kinase M_B with RNase was without marked effect on specific activity of the enzyme.The presence of numerous ribonucleoprotein complexes with a polynucleotide content in the range of 0.5 to 20% and specific activity of 160–220 U/mg protein suggests the control by RNA binding of pyruvate kinase activity from human skeletal muscle.This work was supported by a grant from the Biochemical and Biophysical Committee of the Polish Academy of Sciences.     

Inactivation of maize phosphoenolpyruvate carboxylase by urea

Phosphoenolpyruvate carboxylase purified from leaves of maize (Zea mays, L.) is sensitive to the presence of urea. Exposure to 2.5 m urea for 30 min completely inactivates the enzyme, whereas for a concentration of 1.5 m urea, about 1 h is required. Malate appears to have no effect on inactivation by urea of phosphoenolpyruvate carboxylase. However, the presence of 20 mm phosphoenolpyruvate or 20 mm glucose-6-phosphate prevents significant inactivation by 1.5 m urea for at least 1 h. The inactivation by urea is reversible by dilution. The inhibition by urea and the protective effects of phosphoenolpyruvate and glucose-6-phosphate are associated with changes in aggregation state.     

Effects of Phosphorus Limitation on Respiratory Metabolism in the Green Alga Selenastrum minutum

The effects of phosphorus nutrition on several physiological and biochemical parameters of the green alga, Selenastrum minutum, have been examined. Algal cells were cultured in chemostats under conditions of either Pi limitation or nutrient sufficiency. Pi limitation resulted in: (a) a 5-fold lower rate of respiration, (b) a 3-fold decline in rates of photosynthetic carbon dioxide fixation and oxygen evolution, (c) a 3-fold higher rate of dark carbon dioxide fixation, (d) significant increases in activities of phosphoenolpyruvate (PEP) carboxylase and PEP phosphatase (128% and 158% of nutrient sufficient activities, respectively), (e) significant reductions in activities of nonphosphorylating NADP-glyceraldehyde-3-phosphate dehydrogenase and NAD malic enzyme, and (f) no change in levels of ATP:fructose-6-phosphate 1-phosphotransferase, phosphorylating NAD-glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and pyruvate kinase. The intracellular concentrations of Pi, ATP, AMP, soluble protein, and chlorophyll were also significantly reduced in response to Pi limitation. As well, the level of ADP was about 11-fold lower in the Pi-limited cells as compared to the nutrient sufficient controls. It was predicted that because of this low level of ADP, pyruvate kinase catalyzed conversion of PEP to pyruvate may be restricted in Pi-limited cells. During Pi limitation, PEP carboxylase and PEP phosphatase may function to “bypass” the ADP dependent pyruvate kinase, as well as to recycle Pi for its reassimilation into cellular metabolism.     

New Insights on the Mechanism of the K+-Independent Activity of Crenarchaeota Pyruvate Kinases

Eukarya pyruvate kinases have glutamate at position 117 (numbered according to the rabbit muscle enzyme), whereas in Bacteria have either glutamate or lysine and in Archaea have other residues. Glutamate at this position makes pyruvate kinases K⁺-dependent, whereas lysine confers K⁺-independence because the positively charged residue substitutes for the monovalent cation charge. Interestingly, pyruvate kinases from two characterized Crenarchaeota exhibit K⁺-independent activity, despite having serine at the equivalent position. To better understand pyruvate kinase catalytic activity in the absence of K⁺ or an internal positive charge, the Thermofilum pendens pyruvate kinase (valine at the equivalent position) was characterized. The enzyme activity was K⁺-independent. The kinetic mechanism was random order with a rapid equilibrium, which is equal to the mechanism of the rabbit muscle enzyme in the presence of K⁺ or the mutant E117K in the absence of K⁺. Thus, the substrate binding order of the T. pendens enzyme was independent despite lacking an internal positive charge. Thermal stability studies of this enzyme showed two calorimetric transitions, one attributable to the A and C domains (T_m of 99.2°C), and the other (T_m of 105.2°C) associated with the B domain. In contrast, the rabbit muscle enzyme exhibits a single calorimetric transition (T_m of 65.2°C). The calorimetric and kinetic data indicate that the B domain of this hyperthermophilic enzyme is more stable than the rest of the protein with a conformation that induces the catalytic readiness of the enzyme. B domain interactions of pyruvate kinases that have been determined in Pyrobaculum aerophilum and modeled in T. pendens were compared with those of the rabbit muscle enzyme. The results show that intra- and interdomain interactions of the Crenarchaeota enzymes may account for their higher B domain stability. Thus the structural arrangement of the T. pendens pyruvate kinase could allow charge-independent catalysis.     

Properties of pyruvate kinase from soybean nodule cytosol

The properties of pyruvate kinase from soybean (Glycine max L.) nodule cytosol were examined to determine what influence the N₂ fixation process might have on this supposed key control enzyme. A crude enzyme preparation was prepared by chromatography of cytosol extract on a diethylaminoethyl-cellulose column. ATP and citrate at 5 mm concentrations inhibited pyruvate kinase 27 and 34%, respectively. Enzyme activation was hyperbolic with respect to both K⁺ and NH₄⁺ concentrations. In the presence of physiological concentrations of K⁺ and high phosphoenolpyruvate (PEP) concentrations, NH₄⁺ inhibited enzyme activity. Comparisons of kinetic parameters (V_max and apparent K_a) for NH₄⁺ and K⁺ with inhibition curves indicated that inhibition was very likely a result of competition of the ions for activation site(s) on the pyruvate kinase. In addition, apparent K_a (monovalent cation) and K_m (PEP) were influenced by PEP and monovalent cation concentrations, respectively. This effect may reflect a fundamental difference between plant and animal pyruvate kinases. It is concluded that control of cytosol pyruvate kinase may be closely related to reactions involved in the assimilation of NH₄⁺.     

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.     

Pyruvate Kinase of Streptococcus lactis

The kinetic properties of pyruvate kinase (ATP:pyruvate-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis have been investigated. Positive homotropic kinetics were observed with phosphoenolpyruvate and adenosine 5′-diphosphate, resulting in a sigmoid relationship between reaction velocity and substrate concentrations. This relationship was abolished with an excess of the heterotropic effector fructose-1,6-diphosphate, giving a typical Michaelis-Menten relationship. Increasing the concentration of fructose-1,6-diphosphate increased the apparent V_max values and decreased the K_m values for both substrates. Catalysis by pyruvate kinase proceeded optimally at pH 6.9 to 7.5 and was markedly inhibited by inorganic phosphate and sulfate ions. Under certain conditions adenosine 5′-triphosphate also caused inhibition. The K_m values for phosphoenolpyruvate and adenosine 5′-diphosphate in the presence of 2 mM fructose-1,6-diphosphate were 0.17 mM and 1 mM, respectively. The concentration of fructose-1,6-diphosphate giving one-half maximal velocity with 2 mM phosphoenolpyruvate and 5 mM adenosine 5′-diphosphate was 0.07 mM. The intracellular concentrations of these metabolites (0.8 mM phosphoenolpyruvate, 2.4 mM adenosine 5′-diphosphate, and 18 mM fructose-1,6-diphosphate) suggest that the pyruvate kinase in S. lactis approaches maximal activity in exponentially growing cells. The role of pyruvate kinase in the regulation of the glycolytic pathway in lactic streptococci is discussed.     

Purification of porcine liver pyruvate dehydrogenase complex and characterization of its catalytic and regulatory properties.

Procedures are described for isolating highly purified porcine liver pyruvate and α-ketoglutarate dehydrogenase complexes. Rabbit serum stabilized these enzyme complexes in mitochondrial extracts, apparently by inhibiting lysosomal proteases. The complexes were purified by a three-step procedure involving fractionation with polyethylene glycol, pelleting through 12.5% sucrose, and a second fractionation under altered conditions with polyethylene glycol. Sedimentation equilibrium studies gave a molecular weight of 7.2 × 10⁶ for the liver pyruvate dehydrogenase complex. Kinetic parameters are presented for the reaction catalyzed by the pyruvate dehydrogenase complex and for the regulatory reactions catalyzed by the pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase. For the overall catalytic reaction, the competitive K_i to K_m ratio for NADH versus NAD⁺ and acetyl CoA versus CoA were 4.7 and 5.2, respectively. Near maximal stimulations of pyruvate dehydrogenase kinase by NADH and acetyl CoA were observed at NADH:NAD⁺ and acetyl CoA:CoA ratios of 0.15 and 0.5, respectively. The much lower ratios required for enhanced inactivation of the complex by pyruvate dehydrogenase kinase than for product inhibition indicate that the level of activity of the regulatory enzyme is not directly determined by the relative affinity of substrates and products of catalytic sites in the pyruvate dehydrogenase complex. In the pyruvate dehydrogenase kinase reaction, K⁺ and NH⁺₄ decreased the K_m for ATP and the competitive inhibition constants for ADP and (β,γ-methylene)adenosine triphosphate. Thiamine pyrophosphate strongly inhibited kinase activity. A high concentration of ADP did not alter the degree of inhibition by thiamine pyrophosphate nor did it increase the concentration of thiamine pyrophosphate required for half-maximal inhibition.     

Canine erythrocyte pyruvate kinase. II. Properties of the abnormal enzyme associated with hemolytic anemia in the basenji dog

We have compared the solubility, kinetic, immunological, and electrophoretic properties of erythrocyte pyruvate kinase from normal dogs and Basenji dogs with congenital hemolytic anemia due to pyruvate kinase deficiency. Differences can be detected between the two enzymes by all methods. The enzyme from the affected animals has a greater solubility in ammonium sulfate. It has a lower K _m for phosphoenolpyruvate, while the K _m for ADP is increased. This enzyme is not inhibited by ATP or activated by fructose 1,6-diphosphate. The enzyme from the affected animals has none of the allosteric properties characteristic of the normal canine enzyme. No difference can be detected by enzyme inactivation with rabbit antiserum against the human erythrocyte enzyme, but a slight spur is observed on comparison of the two enzymes by Ouchterlony immunodiffusion. The enzymes also differ in their electrophoretic mobilities on starch gel electrophoresis.     

Reciprocal Regulation of Protein Kinase and Pyruvate Kinase Activities of Pyruvate Kinase M2 by Growth Signals

Pyruvate kinase isoform M2 (PKM2) is an enzyme-catalyzing conversion of phosphoenolpyruvate to pyruvate in the glycolysis pathway. It was demonstrated that PKM2 interacts with tyrosine phosphopeptide, and the interaction with the tyrosine phosphopeptide affects the pyruvate kinase activity of PKM2. Our experiments suggest that PKM2 is also an active protein kinase (Gao, X., Wang, H., Yang, J. J., Liu, X., and Liu, Z. R. (2012) Mol. Cell 45, 598–609). We report here that growth signals reciprocally regulate the pyruvate kinase and protein kinase activities of PKM2 by different mechanisms. On the one hand, growth signals induce protein tyrosine phosphorylations. The tyrosine-phosphorylated protein(s) regulates the conversion of pyruvate kinase and protein kinase of PKM2 by directly interacting with PKM2. Binding of the tyrosyl-phosphorylated proteins at the fructose 1,6-bisphosphate-binding site converts the tetrameric PKM2 to a dimer. On the other hand, growth stimulations also lead to PKM2 phosphorylation, which consequently regulates the conversion of protein kinase and pyruvate kinase activities. Growth factor stimulations significantly increase the dimer/tetramer PKM2 ratio in cells and consequently activate the protein kinase activity of PKM2. Our study suggests that the conversion between the pyruvate kinase and protein kinase activities of PKM2 may be an important mechanism mediating the effects of growth signals in promoting cell proliferation.     

NO(3) Enhances the Kinase Activity for Phosphorylation of Phosphoenolpyruvate Carboxylase and Sucrose Phosphate Synthase Proteins in Wheat Leaves: Evidence from the Effects of Mannose and Okadaic Acid

The aim of this work was to determine which of the two reactions (i.e. phosphorylation or dephosphorylation) involved in the establishment of the phosphorylated status of the wheat leaf phosphoenolpyruvate carboxylase and sucrose phosphate synthase protein responds in vivo to NO₃⁻ uptake and assimilation. Detached mature leaves of wheat (Triticum aestivum L. cv Fidel) were fed with N-free (low-NO₃⁻ leaves) or 40 mm NO₃⁻ solution (high-NO₃⁻ leaves). The specific inhibition of the enzyme-protein kinase or phosphatase activities was obtained in vivo by addition of mannose or okadaic acid, respectively, in the uptake solution. Mannose at 50 mm, by blocking the kinase reaction, inhibited the processes of NO₃⁻-dependent phosphoenolpyruvate carboxylase activation and sucrose phosphate synthase deactivation. Following the addition of mannose, the deactivation of phosphoenolpyruvate carboxylase and the activation of sucrose phosphate synthase, both due to the enzyme-protein dephosphorylation, were at the same rate in low-NO₃⁻ and high-NO₃⁻ leaves, indicating that NO₃⁻ had no effect per se on the enzyme-protein phosphatase activity. Upon treatment with okadaic acid, the higher increase of phosphoenolpyruvate carboxylase and decrease of sucrose phosphate synthase activities observed in high NO₃⁻ compared with low NO₃⁻ leaves showed evidence that NO₃⁻ enhanced the protein kinase activity. These results support the concept that NO₃⁻, or a product of its metabolism, favors the activation of phosphoenolpyruvate carboxylase and deactivation of sucrose phosphate synthase in wheat leaves by promoting the light activation of the enzyme-protein kinase(s) without affecting the phosphatase(s).     

Protein kinases in brown adipose tissue of developing rats II. Two soluble kinase activities and their affinities for nucleotide and protein substrates

A heat-stable, soluble component of brown adipose tissue from newborn rats was found to be readily phosphorylated by protein kinase of the same subcellular fraction. The concentration of this component in brown fat decreased with the age of the animals. A boiled crude microsomal preparation from rat liver was also phosphorylated by brown fat protein kinase. The GTP-linked phosphorylation of the endogenous heat-stable protein was not stimulated by ATP (in contrast to phosphorylation of histone). The maximum velocity of phosphorylation achieved with GTP was about 2.5 times higher than that with ATP as nucleotide substrate. This difference was not due to ATPase activity in the assay. With histone as the protein acceptor both activities were the same. The affinity of protein kinase(s) for ATP was lower with the endogenous heat-stable brown-fat protein and with boiled microsomes (K_m of 0.21 mM and 0.17 mM, respectively) than with histone (K_m of 0.05 M). No detecable ATPase activity was present in either acceptor protein. It is concluded that the 100 000 × g supernatant fraction from brown fat of infant rats contains two protein kinase activities. One preferentially uses ATP and histone as substrates and the other uses endogenous heat-stable soluble proteins and either ATP or GTP.     

Characterization and kinetics of isoenzymes of pyruvate kinase from developing castor bean endosperm

Isozymes of pyruvate kinase (PK) have been isolated from developing castor bean endosperm. One isozyme, PK_c, is localized in the cytosol, and the other, PK_p, is in the plastid. Both isozymes need monovalent and divalent cations for activity, requirements which can be filled by K⁺ and Mg²⁺. Both isozymes are inhibited by citrate, pyruvate, and ATP. PK_c has a much broader pH profile than PK_p and is also more stable. Both have the same K_m (0.05 millimolar) for PEP, but PK_p has a 10-fold higher K_m (0.3 millimolar) for ADP than PK_c (0.03 millimolar). PK_c also has a higher affinity for alternate nucleotide substrates than PK_p. The two isozymes have different kinetic mechanisms. Both have an ordered sequential mechanism and bind phosphoenolpyruvate before ADP. However, the plastid isozyme releases ATP first, whereas pyruvate is the first product released from the cytosolic enzyme. The properties of the two isozymes are similar to those of their counterparts in green tissue.     

Lipid peroxidation and the degradation of cytochrome P-450 heme

The enzyme content and functional capacities of mesophyll chloroplasts from Atriplex spongiosa and maize have been investigated. Accompanying evidence from graded sequential blending of leaves confirmed that mesophyll cells contain all of the leaf pyruvate, P_i dikinase, and PEP carboxylase activities and a major part of the adenylate kinase and pyrophosphatase. 3-Phosphoglycerate kinase, NADP glyceraldehyde-3-P-dehydrogenase, and triose-P isomerase activities were about equally distributed between mesophyll and bundle sheath cells but other Calvin cycle enzymes were very largely or solely located in bundle sheath cells. In A. spongiosa extracts of predominantly mesophyll origin the proportion of the released pyruvate, P_i dikinase, adenylate kinase, pyrophosphatase, 3-phosphoglycerate kinase, and NADP glyceraldehyde-3-P dehydrogenase retained in pelleted chloroplasts was similar but varied between 30 and 80% in different preparations. The proportion of these enzymes and NADP malate dehydrogenase recovered in maize chloroplast preparations varied between 15 and 35%. Washed chloroplasts retained most of the activity of these enzymes but ribulose diphosphate carboxylase and other Calvin cycle enzyme activities were undetectable. Among the evidence for the integrity of these chloroplasts was their capacity for light-dependent conversion of pyruvate to phosphoenolpyruvate and O₂ evolution when 3-phosphoglycerate or oxaloacetate were added. These results support our previous conclusions about the function of mesophyll chloroplasts in C₄-pathway photosynthesis and clearly demonstrate that they lack Calvin cycle activity.     

Partial purification from hepatoma cells of an intracellular substance which mediates the effects of insulin on pyruvate dehydrogenase and low Km cyclic AMP phosphodiesterase

A substance capable of stimulating the activities of pyruvate dehydrogenase and low K_m cyclic AMP phosphodiesterase was prepared from H4-II-EC3′ hepatoma cells by acid extraction and partially purified by molecular exclusion chromatography. The material thus prepared by gel chromatography was found to stimulate the activities of these enzymes in a concentration-dependent manner. The amount or activity of the pyruvate dehydrogenase stimulating factor was increased in cells which had been treated with physiological concentrations of insulin (0.2 mU/ml). Increasing the concentration of insulin increased the amount or activity of the factor generated. High concentrations of insulin did not cause a reversal of the effects of insulin. The stimulation of pyruvate dehydrogenase activity by the factor was eliminated when sodium fluoride (75 mm) was present in the enzyme assay, implying that activation was mediated by the pyruvate dehydrogenase phosphatase. The enzyme-stimulating factor isolated from hepatoma cells shares a number of important characteristics with the putative second messenger of insulin prepared from other cell types: (1) it is heat and acid stable, (2) it has a similar apparent molecular weight, (3) it is generated in an insulin-dependent manner, (4) it stimulates the activity of pyruvate dehydrogenase by a fluoride-sensitive mechanism, and (5) it elutes from the anion-exchange resin AG 1-X8 at an ionic strength of 0.4 m. These findings suggest that the stimulator of pyruvate dehydrogenase and of low K_m cyclic AMP phosphodiesterase isolated from hepatoma cells has chemical properties identical with those of the putative second messenger of insulin action isolated from a number of other insulin-sensitive tissues.     

Phosphate Deficiency in Maize : III. Changes in Enzyme Activities during the Course of Phosphate Deprivation

The effects of low concentrations of phosphate (low-P) on soluble protein content, the activities of 12 different enzymes, and the rates of photosynthesis and respiration on the basis of leaf area were investigated in maize (Zea mays L.) leaves 16 to 24 days after planting (DAP). With low-P treatment, a drastic decrease in the rate of photosynthesis to only 6% of the maximum rate in control plants was observed by 24 DAP. Low-P treatment had almost no effect on the rate of respiration until 21 DAP, but then the rate of respiration decreased progressively to about 55% of the maximum rate in control plants. The soluble protein content in low-P plants decreased to 56% of the maximum content in control plants. The changes in the activities of enzymes in low-P plants showed several different patterns. The activities of pyruvate, orthophosphate dikinase, 3-phosphoglycerate kinase, phosphoenolpyruvate carboxylase (PEPC), ribulose 1,5-bisphosphate carboxylase, fructose 1,6-bisphosphate aldolase, catalase, phosphohexose isomerase, chloroplastic fructose 1,6-bisphosphatase, and ADP-glucose-pyrophosphorylase decreased steadily from 85 to 100% of the maximum activity found in 18- to 21-day-old control plants (V_max) to 30 to 70% of V_max. The activity of sucrose phosphate synthase remained virtually constant at approximately 85 to 100% of V_max. The activity of UDP-glucose-pyrophosphorylase remained almost constant up to 21 DAP and then decreased to 80% of V_max by 24 DAP. The activity of cytochrome c oxidase increased slightly up to 21 DAP but then decreased to 50% of V_max by 24 DAP. As indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of soluble proteins, the subunit of PEPC stained less intensely in 24-d-old low-P plants. The possibility is discussed that during low-P treatment there is selective degradation of PEPC without a concomitant degradation of sucrose phosphate synthase, both of which are known to be localized in the cytoplasmic compartment of mesophyll cells.     

The effects of calcium ions on the activities of trehalase, hexokinase, phosphofructokinase, fructose diphosphatase and pyruvate kinase from various muscles

1. The effects of Ca²⁺ on the activities and regulatory properties of trehalase, hexokinase, phosphofructokinase, fructose diphosphatase and pyruvate kinase from vertebrate red and white muscle and insect fibrillar and non-fibrillar muscle have been investigated. These muscles were selected because of the possible difference in the role of glycolysis in energy production in the vertebrate muscles, and the possible difference in the role of Ca²⁺ in the control of contraction in the two types of insect muscle. An increase in Ca²⁺ concentration from 0.001μm to 10μm did not modify the activities nor did it modify the regulatory properties of these enzymes from these various muscles. 2. Concentrations of Ca²⁺ above 0.1mm inhibited the activities of hexokinase and phosphofructokinase from the different muscles. It has been suggested that this inhibition may provide the basis for a theory of regulation of glycolysis (Margreth et al., 1967). If phosphofructokinase is located within the sarcoplasmic reticulum, its activity will be inhibited when the muscle is at rest, but the release of Ca²⁺ from the reticulum during contraction will lead to a stimulation of its activity and hence an increase in glycolytic flux. The distribution of hexokinase and phosphofructokinase in the various cell fractions of these muscles was very variable. In particular, both enzymes were present almost exclusively in the 100000g supernatant fraction in the extracts of insect flight muscles. Thus there is no correlation between the properties of the enzymes and their distribution in muscle. 3. It is concluded that Ca²⁺ does not control the activities of the important regulatory enzymes of glycolysis in muscle. It is suggested that in some muscles the sensitivity of the control mechanism at the level of phosphofructokinase to changes in the concentration of AMP may be increased by a process known as `substrate-cycling'.     

Functional analysis, overexpression, and kinetic characterization of pyruvate kinase from Plasmodium falciparum

The important role of pyruvate kinase during malarial infection has prompted the cloning of a cDNA encoding Plasmodium falciparum pyruvate kinase (pfPyrK), using mRNA from intraerythrocytic-stage malaria parasites. The full-length cDNA encodes a protein with a computed molecular weight of 55.6 kDa and an isoelectric point of 7.5. The purified recombinant pfPyrK is enzymatically active and exists as a homotetramer in its active form. The enzyme exhibits hyperbolic kinetics with respect to phosphoenolpyruvate and ADP, with K_m of 0.19 and 0.12 mM, respectively. pfPyrK is not affected by fructose-1,6-bisphosphate, a general activating factor of pyruvate kinase for most species. Glucose-6-phosphate, an activator of the Toxoplasma gondii enzyme, does not affect pfPyrK activity. Similar to rabbit pyruvate kinase, pfPyrK is susceptible to inactivation by 1 mM pyridoxal-5′-phosphate, but to a lesser extent. A screen for inhibitors to pfPyrK revealed that it is markedly inhibited by ATP and citrate. Detailed kinetic analysis revealed a transition from hyperbolic to sigmoidal kinetics for PEP in the presence of citrate, as well as competitive inhibitory behavior for ATP with respect to PEP. Citrate exhibits non-competitive inhibition with respect to ADP with a K_i of 0.8 mM. In conclusion, P. falciparum expresses an active pyruvate kinase during the intraerythrocytic-stage of its developmental cycle that may play important metabolic roles during infection.