A simple procedure for the synthesis of [32P]phosphoenolpyruvate via the pyruvate kinase exchange reaction at equilibrium

A one step procedure is presented for the preparation of [³²P]phosphoenolpyruvate from [γ-³²P]ATP using pyruvate kinase. The reaction is carried out at chemical equilibrium and involves only an exchange of isotope between ATP and phosphoenolpyruvate. The initial phosphoenolpyruvate/ATP ratio in the reaction mixture determines the degree of ³²P incorporation into phosphoenolpyruvate when isotopic equilibrium is achieved.     

Regulation of hepatic phosphoenolpyruvate carboxykinase (GTP) Role of dietary proteins and amino acids in vivo and in the isolated perfused rat liver

The effect of protein feeding and the addition of amino acids on the activity of hepatic phosphoenolpyruvate carboxykinase (GTP: oxalacetate carboxylyase (transphosphorylating), EC 4.1.1.32) was investigated in vivo and in the isolated perfused rat liver. Protein feeding resulted in a considerable increase in phosphoenolpyruvate carboxykinase activity within 6 h. This rise was independent of the presence of glucocorticoids.In the isolated perfused liver system amino acids per se had a small effect on phosphoenolpyruvate carboxykinase activity and led to an increase by 20% when glucocorticoids were present, but resulted in a rise by 100% when glucocorticoids plus dibutyryl cyclic AMP were added to the perfusion medium. The effect of amino acids in the presence of dibutyryl cyclic AMP could also be observed in the liver of glucocorticoid-deprived rats.Cycloheximide, a translational inhibitor, totally blocked all effects of amino acids on enzyme activity.These results indicate that the concentration of amino acids in the portal vein modify the regulation of phosphoenolpyruvate carboxykinase by cyclic AMP.     

Pyruvate cycling involving possible oxaloacetate decarboxylase activity

With high concentrations of pyruvate as substrate for hepatocytes from fasted rats, high rates of cycling between pyruvate and the dicarboxylic acids occur, as shown isotopically. This rate of cycling is adequate to account for the hydrogen translocation from the mitochondria to the cytosol to furnish NADH for lactate formation. Addition of sufficiently high concentrations of mercaptopicolinate to block almost completely glucose formation from pyruvate, depresses isotopic cycling and lactate formation by only about 50–75%. Under some conditions, when the normal phosphoenolpyruvate carboxykinase activity is inhibited, cytosolic oxaloacetate may be decarboxylated directly to pyruvate, possibly via the decarboxylase activity of phosphoenolpyruvate carboxykinase.     

The disposition of citric acid cycle intermediates by isolated rat heart mitochondria

The mechanism of depletion of tricarboxylic acid cycle intermediates by isolated rat heart mitochondria was studied using hydroxymalonate (an inhibitor of malic enzymes) and mercaptopicolinate (an inhibitor of phosphoenolpyruvate carboxykinase) as tools. Hydroxymalonate inhibited the respiration rate of isolated mitochondria in state 3 by 40% when 2 mM malate was the only external substrate, but no inhibition was found with 2 mM malate plus 0.5 mM pyruvate as substrates. In the prescence od bicarbonate, arsenite and ATP, propionate was converted to pyruvate and malate at the rates of 14.0 ± 2.9 and 2.8 ± 1.8 nmol/mg protein in 5 min, respectively. Under these conditions, 0.1 mM mercaptopicolinate did not affect this conversion, but 2 mM hydroxymalonate inhibited pyruvate formation completely and resulted in an accumulation of malate up to 13.2 ± 2.9 nmol/mg protein. No accumulation of phosphoenolpyruvate was found under any condition tested. It is concluded that malic enzymes but not phosphoenolpyruvate carboxykinase, are involved in conversion of propionate to pyruvate in isolated rat heart mitochondria.     

Glutamate metabolism in relation to glutamate transport in kidney cortex mitochondria of rabbit

1. The metabolism of glutamate was followed by measurements of phosphoenolpyruvate production, aspartate synthesis and ammonia release, whereas the transport of glutamate across the inner membrane of kidney cortex mitochondria was studied using an oxygen electrode and the swelling technique.2. When added separately, avenaciolide and aminooxyacetate only partially inhibited both State 3 and uncoupled respiration of the mitochondria, as studied in the presence of glutamate as substrate. In contrast, the addition of both inhibitors to the reaction medium resulted in an almost complete inhibition of glutamate oxidation.3. Swelling of kidney mitochondria in an isosmotic solution of ammonium glutamate was accelerated by uncoupler and inhibited by avenaciolide, while the swelling of mitochondria in potassium glutamate was stimulated by valinomycin and inhibited by uncoupler.4. When glutamate was used as the sole substrate, inhibition of aspartate formation by aminooxyacetate resulted in a stimulation of both ammonia release and phosphoenolpyruvate production. In contrast, with glutamate plus malate as substrate an elevation of the rate of glutamate deamination on the addition of aminooxyacetate was accompanied by an inhibition of phosphoenolpyruvate synthesis in both State 3 and uncoupled conditions.5. In the presence of valinomycin to induce K⁺-permeability a marked enhancement of glutamate deamination was accompanied by a significant inhibition of glutamate transamination.6. Based on the presented results it was concluded that in rabbit renal mitochondria utilizing glutamate as substrate the rates of ammonia production, phosphoenolpyruvate formation and aspartate synthesis vary in response to different metabolic conditions, in which both the glutamate—H⁺ symport and the glutamate—aspartate exchange systems are functioning to different extents.     

Light Moderates the Induction of Phosphoenolpyruvate Carboxylase by NaCl and Abscisic Acid in Mesembryanthemum crystallinum

In Mesembryanthemum crystallinum, phosphoenolpyruvate carboxylase is synthesized de novo in response to osmotic stress, as part of the switch from C₃-photosynthesis to Crassulacean acid metabolism. To better understand the environmental signals involved in this pathway, we have investigated the effects of light on the induced expression of phosphoenolpyruvate carboxylase mRNA and protein in response to stress by 400 millimolar NaCl or 10 micromolar abscisic acid in hydroponically grown plants. When plants were grown in high-intensity fluorescent or incandescent light (850 microeinsteins per square meter per second), NaCl and abscisic acid induced approximately an eightfold accumulation of phosphoenolpyruvate carboxylase mRNA when compared to untreated controls. Levels of phosphoenolpyruvate carboxylase protein were high in these abscisic acid- and NaCl-treated plants, and detectable in the unstressed control. Growth in high-intensity incandescent (red) light resulted in approximately twofold higher levels of phosphoenolpyruvate carboxylase mRNA in the untreated plants when compared to control plants grown in high-intensity fluorescent light. In low light (300 microeinsteins per square meter per second fluorescent), only NaCl induced mRNA levels significantly above the untreated controls. Low light grown abscisic acid- and NaCl-treated plants contained a small amount of phosphoenolpyruvate carboxylase protein, whereas the (untreated) control plants did not contain detectable amounts of phosphoenolpyruvate carboxylase. Environmental stimuli, such as light and osmotic stress, exert a combined effect on gene expression in this facultative halophyte.     

Fluorescence Study of Chemical Modification of Phosphoenolpyruvate Carboxylase from Crassula argentea

The chemical modification of phosphoenolpyruvate carboxylase purified from Crassula argentea leaves was studied using the fluorescence of the extrinsic probe 8-anilino-1-naphalenesulfonate. The effects of ligands on kinetic parameters of phosphoenolpyruvate carboxylase activity, and its response to pH and metal cations, were associated with the binding of the ligands to the enzyme as measured by fluorescence. Binding of the ligands phosphoenolpyruvate, malate, and glucose-6-phosphate revealed by fluorescence measurements corresponds to competitive phenomena observed in kinetic studies. The fluorescence measurements also suggest the involvement of specific amino acids in the binding of a given ligand. Arginyl residues modified by 2,3-butanedione appear to be directly involved in the binding of phosphoenolpyruvate and malate to the active and the inhibition sites, respectively. A histidyl residue was involved in the binding of malate, accounting for the lack of inhibition by malate in kinetic studies of the enzyme treated with diethylpyrocarbonate. Although activity was lost, there was no decrease in the ability of the treated enzyme to bind phosphoenolpyruvate, suggesting that additional histidyl residues are essential for activity although not directly involved in the binding of phosphoenolpyruvate. The lysine reagent trinitrobenzenesulfonate caused a loss of activity and a reduction in malate inhibition and glucose-6-phosphate activation, but these modifications were not related to changes in the ability of the enzyme to bind any of the three ligands. This suggests that lysine residues were not directly involved in the binding of these ligands.     

Purification and Characterization of a Phosphoenolpyruvate Phosphatase from Brassica nigra Suspension Cells

Phosphoenolpyruvate phosphatase from Brassica nigra leaf petiole suspension cells has been purified 1700-fold to apparent homogeneity and a final specific activity of 380 micromole pyruvate produced per minute per milligram protein. Purification steps included: ammonium sulfate fractionation, S-Sepharose, chelating Sepharose, concanavalin A Sepharose, and Superose 12 chromatography. The native protein was monomeric with a molecular mass of 56 kilodaltons as estimated by analytical gel filtration. The enzyme displayed a broad pH optimum of about pH 5.6 and was relatively heat stable. Western blots of microgram quantities of the final preparation showed no cross-reactivity when probed with rabbit polyclonal antibodies prepared against either castor bean endosperm cytosolic pyruvate kinase, or sorghum leaf phosphoenolpyruvate carboxylase. The final preparation exhibited a broad substrate selectivity, showing high activity toward p-nitrophenyl phosphate, adenosine diphosphate, adenosine triphosphate, gluconate 6-phosphate, and phosphoenolpyruvate, and moderate activity toward several other organic phosphates. Phosphoenolpyruvate phosphatase possessed at least a fivefold and sixfold greater affinity and specificity constant, respectively, for phosphoenolpyruvate (apparent Michaelis constant = 50 micromolar) than for any other nonartificial substrate. The enzyme was activated 1.7-fold by 4 millimolar magnesium, but was strongly inhibited by molybdate, fluoride, zinc, copper, iron, and lead ions, as well as by orthophosphate, ascorbate, glutamate, aspartate, and various organic phosphate compounds. It is postulated that phosphoenolpyruvate phosphatase functions to bypass the adenosine diphosphate dependent pyruvate kinase reaction during extended periods of orthophosphate starvation.     

Effect of insulin and prednisolone on cyclic nucleotides and phosphoenolpyruvate carboxykinase activity in brown fat and liver of developing rats

The concentrations of cyclic AMP and cyclic GMP in brown fat and liver of both suckling and adult rats at fixed times after injection of insulin (2.5 U/100 g body weight) or prednisolone (2.5 mg/100 g body weight) were compared with the activity of phosphoenolpyruvate carboxykinase assayed 24 h after the injections. A stimulus that produced an increase in cyclic AMP content also produced an increase in the enzyme activity. If the content of cyclic GMP was also increased there was no rise in phosphenolpyruvate carboxykinase activity. A rise in the content of cyclic GMP alone was associated with a reduction in the activity of the enzyme. These preliminary results indicate that cyclic AMP could be involved in the induction of phosphenolpyruvate carboxykinase and that cyclic GMP may somehow be related to its repression. The known differences in the response of phosphenolpyruvate carboxykinase activity to insulin and prednisolone in different tissues and at different stages of ontogenic development may thus be linked to differences in the responsiveness of enzymes concerned with the metabolism of cyclic nucleotides.     

Isolation and characterization of the tricarboxylate transporter from pea mitochondria

The tricarboxylate transporter was solubilized from pea (Pisum sativum) mitochondria with Triton X-114, partially purified over a hydroxylapatite column, and reconstituted in phospholipid vesicles. The proteoliposomes exchanged external [¹⁴C]citrate for internal citrate or malate but not for preloaded d,l-isocitrate. Similarly, although external malate, succinate, and citrate competed with [¹⁴C]citrate in the exchange reaction, d,l-isocitrate and phosphoenolpyruvate did not. This tricarboxylate transporter differed from the equivalent activity from animal tissues in that it did not transport isocitrate and phosphoenolpyruvate. In addition, tricarboxylate transport in isolated plant mitochondria, as well as that measured with the partially purified and reconstituted transporter, was less active than the transporter isolated from animal tissues.     

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.     

Contribution of Malic Enzyme, Pyruvate Kinase, Phosphoenolpyruvate Carboxylase, and the Krebs Cycle to Respiration and Biosynthesis and to Intracellular pH Regulation during Hypoxia in Maize Root Tips Observed by Nuclear Magnetic Resonance Imaging and Gas Chromatography-Mass Spectrometry

In vivo pyruvate synthesis by malic enzyme (ME) and pyruvate kinase and in vivo malate synthesis by phosphoenolpyruvate carboxylase and the Krebs cycle were measured by ¹³C incorporation from [1-¹³C]glucose into glucose-6-phosphate, alanine, glutamate, aspartate, and malate. These metabolites were isolated from maize (Zea mays L.) root tips under aerobic and hypoxic conditions. ¹³C-Nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry were used to discern the positional isotopic distribution within each metabolite. This information was applied to a simple precursor-product model that enabled calculation of specific metabolic fluxes. In respiring root tips, ME was found to contribute only approximately 3% of the pyruvate synthesized, whereas pyruvate kinase contributed the balance. The activity of ME increased greater than 6-fold early in hypoxia, and then declined coincident with depletion of cytosolic malate and aspartate. We found that in respiring root tips, anaplerotic phosphoenolpyruvate carboxylase activity was high relative to ME, and therefore did not limit synthesis of pyruvate by ME. The significance of in vivo pyruvate synthesis by ME is discussed with respect to malate and pyruvate utilization by isolated mitochondria and intracellular pH regulation under hypoxia.     

deltaC Values in Maize Leaf Correlate with Phosphoenolpyruvate Carboxylase Levels

Values of δ¹³C and levels of phosphoenolpyruvate carboxylase and ribulose 1,5-bisphosphate carboxylase/oxygenase were analyzed in segments from the fourth leaf of young maize (Zea mays L.) plants. The δ¹³C values became significantly more negative from the base to the tip of the leaves. Phosphoenolpyruvate carboxylase levels and ribulose bisphosphate carboxylase levels both increased from the base to the tip. The principal effect of phosphoenolpyruvate carboxylase levels or δ¹³C should arise through its effect on the carboxylation/diffusion balance in the mesophyll. In this case, δ¹³C values should become more negative as phosphoenolpyruvate carboxylase levels increase, unless there are offsetting changes in stomatal aperture. The principal effect of ribulose bisphosphate carboxylase/oxygenase on δ¹³C should occur through its effect on the extent of leakage of CO₂ from the bundle sheath cells. In this case, δ¹³C values should become more positive as ribulose bisphosphate carboxylase levels increase. Accordingly, the variation in δ¹³C values seen in maize leaves appears to be the result of variations in the level of phosphoenolpyruvate carboxylase.     

Unmasking of an essential thiol during function of the membrane-bound enzyme II of the phosphoenolpyruvate β-glucoside phosphotransferase system of Escherichia coli

β-Glucoside transport by phosphoenolpyruvate-hexose phosphotransferase system in Escherichia coli is inactivated in vivo by thiol reagents. This inactivation is strongly enhanced by the presence of transported substrates. In a system reconstituted from soluble and membrane-bound components, only the particulate component, the membrane-bound enzyme II^bgl appeared as the target of N-ethylmaleimide inactivation. The same feature was found in the case of methyl-α-d-glucoside uptake via enzyme II^glc.It is shown that the sensitizing effect of substrates is specific and not generalized, methyl-α-d-glucoside only sensitizes enzyme II^bglc and $\text{p-}\text{nitrophenyl-β-}\text{d}\text{-glucoside}$ only sensitizes enzyme II^bgl towards N-ethylmaleimide inactivation.The inactivation of enzyme II^bgl by thiol reagents is also promoted in vivo by fluoride inhibition of phosphoenolpyruvate synthesis. In toluene-treated bacteria, the presence of phosphoenolpyruvate protects against inactivation by thiol reagents of $\text{p-}\text{nitrophenyl-β-}\text{d}\text{-glucoside}$ phosphorylation. Both results suggest that the inactivator resistent form of enzyme II^bgl is an energized form of the enzyme.     

Light Induction and the Effect of Nitrogen Status upon the Activity of Carbonic Anhydrase in Maize Leaves

The regulation of carbonic anhydrase (CA) activity in maize (Zea mays L.) leaves by light and nitrogen nutrition was determined. CA activity increased by more than 100-fold in illuminated leaves and decreased in leaves placed in the dark; low levels of CA activity were observed in leaves illuminated with low light intensities. CA activity was reduced in plants grown under nitrogen deficiency and recovered only slowly when supplemented with nitrate. Parallel studies were conducted to follow the levels of phosphoenolpyruvate carboxylase. Experiments indicate that the level of CA and phosphoenolpyruvate carboxylase present in leaves may be controlled by similar mechanisms.     

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.     

Role of cysteine in activation and allosteric regulation of maize phosphoenolpyruvate carboxylase

The effect of 5-5′-dithiobis-2-nitrobenzoate (DTNB) on the kinetic parameters and structure of phosphoenolpyruvate carboxylase purified from maize (Zea mays L.) has been studied. The V_max is found to be independent of the presence of this thiol reagent. The K_m is increased upon oxidation of cysteines by DTNB. At a substrate concentration higher than K_m (3.1 millimolar Mgphosphoenolpyruvate), a significant reversible decrease of the activity is observed. Malate has little effect in preventing the modification of these cysteines. The V type inhibition by malate was also studied at a saturating phosphoenolpyruvate level (9.3 millimolar Mgphosphoenolpyruvate). In the presence of 50 micromolar DTNB, up to 60% inhibition is caused by 15 millimolar malate; however, in the presence of both 50 micromolar DTNB and 50 millimolar dithiothreitol (DTT) this inhibition is reduced to 20%. The presence of DTT alone increases the size of the phosphoenolpyruvate carboxylase molecule as determined by light scattering. The activity at nonsaturating substrate concentration is increased by 36% in the presence of DTT. The oligomerization equilibrium between the dimer and the tetrameric form of the enzyme is affected by cysteine. The K_m for the substrate, the sensitivity toward malate, and the size of the enzyme are found to be modified upon incubation in the presence of DTT.