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PubMed1988

Domain interaction in rabbit muscle pyruvate kinase. I. Effects of ligands on protein denaturation induced by guanidine hydrochloride.

The structural stability of rabbit muscle pyruvate kinase was examined. The unfolding of pyruvate kinase was induced by guanidine hydrochloride, and the process was monitored by spectroscopic techniques (fluorescence and UV absorption) and hydrodynamic measurements (sedimentation velocity, sedimentation equilibrium, densimetry, and viscometry). The spectroscopic techniques revealed that the unfolding of pyruvate kinase induced by guanidine hydrochloride is not a simple cooperative process. This suggests that different regions of pyruvate kinase are unfolding with different efficiencies in response to the denaturant. These regions are most likely related to the domain structures observed by x-ray crystallography. In the presence of L-phenylalanine, the allosteric inhibitor, the denaturation process became more cooperative, and the enzyme dissociated and unfolded at a higher denaturant concentration. The binding of phenylalanine also induced a structural change in the enzyme, rendering it more susceptible to tryptic digestion. One of the peptides, the production rate of which was increased, was isolated and sequenced. Its N terminus is located at the interface between two domains, one of which contains the active site. This evidence indicates structural changes, probably involving domain-domain interaction, for pyruvate kinase in response to phenylalanine binding.

Algorithms↗
PubMedDate not supplied

[Ability of the tissues to assimilate glucose and the pyruvate and lactate levels after intravenous glucose load in patients with peripheral facial paralysis].

In 60 control subjects and 48 patients with peripheral facial nerve palsy carbohydrate metabolism was tested performing a rapid intravenous glucose tolerance test with 0.5 g glucose per 1 kg of actual body weight and with determination of concentrations of glucose, pyruvate and lactate at rest in fasting state and after intravenous glucose load. Glucose was determined in capillary blood by the orthotoluidine method, pyruvate and lactate in venous blood by the enzymatic method. In the group of patients with facial nerve palsy a significantly lower number of subjects had a normal ability of glucose assimilation by tissues and a significantly higher frequency of pathological results of glucose tolerance test with a high prevalence of fasting hyperglycaemia. In patients with abnormal glucose tolerance test the fasting pyruvate concentration was high. After an intravenous glucose load the concentration of pyruvate in the 120th minute of the test was significantly increased (p less than 0.05) in patients with facial nerve palsy independently of the degree of glucose tolerance impairment. In patients with pathological result of the glucose tolerance test disturbances were observed also in lactate concentration. The production of lactate was decreased in the initial phase and then it was increased in the time up to 120 minutes. No direct correlation was observed between the disturbances of pyruvate concentration and the preserved ability of glucose assimilation by the peripheral tissues.

Adult↗
PubMed1985

[Pyruvate dehydrogenase deficiency in a child with persistent lactic acidosis].

Evidence is presented for defective pyruvate dehydrogenase (EC 4.1.1.1) in leukocytes and muscle tissue from a 10-year old child with persistent lactic acidosis, suffering from myasthenia and growth retardation. The defect is expressed in vitro by a depressed stimulation of pyruvate dehydrogenase catalytic activity by exogenous phosphoprotein phosphatase, and in vivo by a lack of response to muscle work, in comparison with healthy controls. Pyruvate dehydrogenase activity is in the normal range when measured without addition of phosphoprotein phosphatase in cells obtained from the resting patient. The defect reported here represents a new, hitherto undescribed form of a pyruvate dehydrogenase deficiency. The insufficient catalytic activity explains the observed accumulation of pyruvate, lactate, oxaloacetate and alanine and the decrease of citrate concentration in the blood of this patient. Electron microscope studies of the muscle tissue show an enhanced number of enlarged mitochondria with bizarre shapes and high densities of cristae.

Acidosis↗
PubMed1986

Regulation of pyruvate kinase in cultured rat hepatocytes. Influence of glucose, ethanol, glucagon, and dexamethasone.

The hormonal regulation and molecular forms of Type L pyruvate kinase were investigated in rat hepatocytes maintained in primary culture. Five isoelectric forms of the enzyme subunit were identified by isoelectric focusing in 8 M urea. Immediately after pulse labeling rat hepatocytes with [35S]methionine radioactivity was observed in one major (D-band) and one minor (I-band) peptide band. These isoelectric forms were shown to be dephosphorylated forms of the subunit. Acute administration of 0.1 microM glucagon was accompanied by disappearance of the D- and I-bands and appearance of two additional forms (P- and A-bands, respectively). These latter two forms were demonstrated to be phosphorylated forms of the subunit. A fifth isoelectric form of the pyruvate kinase subunit (B-band) was identified by immunolocation; however, incorporation of radioisotope into this band was low. Chronic administration of glucagon or dexamethasone had no significant influence on the molecular properties of pyruvate kinase. However, novel observations concerning the influence of glucose and ethanol on the phosphorylation state of the enzyme were made. When hepatocytes were maintained at 5.5 mM glucose for 24-48 h, the activity ratio for pyruvate kinase decreased from 0.65 to 0.40 and the enzyme became partially phosphorylated. Raising the glucose concentration to 28 mM prevented or rapidly reversed the phosphorylation state of the enzyme. Administration of low concentrations of ethanol (1-20 mM) caused a decline in the activity ratio of pyruvate kinase in the presence of both 5.5 and 28 mM glucose. These latter observations concerning the influence of glucose and ethanol are the first demonstrating that nutrients or metabolites alter the phosphorylation state of the enzyme in the absence of hormonal stimuli.

Animals↗
PubMed1978

Biochemical effects of niridazole. II. In vitro and in vivo effects of niridazole on the rate of gluconeogenesis and the rate of oxidation of pyruvate and some Krebs cycle intermediates in Schistosoma mansoni infected mice.

The effects of the antischistosomal drug, niridazole, on the rate of gluconeogenesis in kidney cortex slices and on the rate of oxidation of pyruvate and some Krebs cycle intermediates in liver homogenates of infected mice were described. The effect of schistosoma mansoni infection on the previously mentioned parameters was also described. The infection per se did not affect the rate of gluconeogenesis from pyruvate, succinate and alpha-ketoglutarate when used as gluconeogenic precursors. In case of the rates of oxidation of pyruvate, succinate alpha-ketoglutarate and citrate, the infection decreased them significantly. In vitro, niridazole did not increase the inhibition of the rate of oxidation of different substances studied caused by the infection per se. The rate of gluconeogenesis from alpha-ketoglutarate was also unaffected. In vivo, niridazole did not affect the oxidoreductases more than did the infection per se. In fact in many cases, the drug tended to normalize the inhibitory effect of the infection on some of the enzyme systems, particularly in the case of the citrate succinate and pyruvate. On administration of 100 mg/kg of niridazole for 5 days (i.e. low dosage only) the rate of gluconeogenesis from pyruvate and alpha-ketoglutarate was stimulated. Such effects seem to be related to the presence of metabolites rather than to the parent drug.

Animals↗
PubMed1983

Diminution of stationary enzyme activities at increases of pyruvate kinase concentration in a reconstituted enzyme system.

In a homogeneous and open enzyme system containing phosphofructokinase, pyruvate kinase, adenylate kinase, and glucose 6-phosphate isomerase the consequences of variations of the enzyme concentrations on the stationary enzyme activities have been investigated. An unexpected behavior was observed upon variation of the maximum activity of pyruvate kinase. Depending on the experimental conditions an increase of the concentration of pyruvate kinase resulted either in a diminution or in a stimulation of the stationary activity of this enzyme. An increase of the maximum activity of phosphofructokinase, however, stimulates both the activities of phosphofructokinase and pyruvate kinase. The experimental results are interpreted in terms of a mathematical model, based on the kinetic properties of the enzymes involved. The correlation between the observed changes of the activities of phosphofructokinase and pyruvate kinase and the appearance of multiple stationary states is discussed.

Adenylate Kinase↗
PubMed1983

Regulatory properties of the pyruvate dehydrogenase complex from Escherichia coli. Studies on the thiamin diphosphate-dependent lag phase.

The pyruvate dehydrogenase complex from Escherichia coli shows an appreciable lag phase (tau) of some minutes when its overall reaction rate was tested with very limiting amounts of thiamin diphosphate. tau depends on the concentration of thiamin diphosphate in a nonlinear fashion. Sodium diphosphate, a competitive inhibitor with respect to thiamin diphosphate (Ki = 5.2 . 10(-4) M) prolongs the lag, while the strongly binding transition state analog thiamin thiazolone diphosphate has no effect. tau is independent of the enzyme concentration, thus no dissociation-association step is involved. Incubation of the pyruvate dehydrogenase complex with thiamin diphosphate, Mg2+, and pyruvate leads to a shortening of the lag phase, as well as to a decrease of the intrinsic tryptophan fluorescence in a time-dependent process, which evinces the same characteristics as tau. Dependence of pyruvate, as well as of the substrate analog methylacetylphosphonate, can be established by measurements of fluorescence quenching, thus ruling out an essential role of hydroxyethyl thiamin diphosphate in the process reflected by the lag phase. The results demonstrate that the lag phase is induced after the binding of both thiamin diphosphate . Mg2+ and pyruvate to the catalytic site to form a ternary enzyme complex, which undergoes subsequently a slow conformational change to an active enzyme form. This change is confined to single subunits, and no interactions between neighboring monomers could be observed. A model is proposed to describe the mechanism represented by the lag phase.

Diphosphates↗
PubMed1983

Structural and kinetic differences between the M2 type pyruvate kinases from lung and various tumors.

The kinetic and structural properties of purified, homogeneous pyruvate kinase type M2 from chicken lung and tumors, including that from Rous sarcoma virus-transformed chicken fibroblasts, have been compared. The "tumor enzyme" is characterized by a low affinity for phosphoenolpyruvate, pronounced serine activation, and strong alanine inhibition as compared to the "lung type". In contrast to the rat lung enzyme, which is not affected by serine, the chicken lung enzyme is slightly activated by serine. The serine metabolites phosphoserine and glycine do not activate the "tumor type M2 pyruvate kinase", but L-alpha-glycerophosphorylcholine and phosphatidylserine do slightly activate at physiological concentrations. Studies with substances structurally related to serine reveal that the hydroxyl group of serine is a prerequisite for the activation and that the amino and carboxyl groups determine the affinity of the "tumor type M2 pyruvate kinase" for serine. Two different fragmentation methods (CNBr-cleavage and V-8 proteolysis) and two different methods for separation of the resulting peptide fragments (polyacrylamide gel isoelectric focussing and SDS-polyacrylamide electrophoresis) indicated a high degree of homology between the type M2 pyruvate kinases from lung and tumors as well the type M1 from muscle. Each type of pyruvate kinase, however, contains one or two unique protein fragments which are characteristic for its type. We have termed those fragments L (lung), M (muscle), and T (tumor).

Animals↗
PubMed1983

Regulation of the synthesis and degradation of pyruvate carboxylase in 3T3-L1 cells.

The differentiation of mouse 3T3-L1 cells is characterized by an accumulation of cytosolic triglyceride and marked increase in many enzymatic activities involved in triglyceride biosynthesis. The specific activity of one such enzyme, pyruvate carboxylase, increases at least 20-fold and is due to a parallel increase in the intracellular concentration of the protein. Pulse-labeling experiments demonstrated that the increase in the specific activity of pyruvate carboxylase was due to an increase in the rate of enzyme synthesis. In the differentiated cell, pyruvate carboxylase represented 1.9% of the total cellular protein and 1% of the protein radiolabeled during a 1-h pulse. This was 35-and 28-fold higher than in the undifferentiated cell, respectively. The turnover of pyruvate carboxylase in the differentiated cell was similar to that in the undifferentiated cell with the enzyme having a half-life of 28-35 h. The half-life of apopyruvate carboxylase in avidin-treated 3T3-L1 cells was 24 h, indicating that the turnover of the apoenzyme was not significantly different than that of the holoenzyme. Radiolabeling pyruvate carboxylase with [14C]biotin and [3H]leucine demonstrated that the turnover of biotin associated with the enzyme was identical to the turnover of the enzymatic protein.

Animals↗
PubMed1983

The catalytic mechanism of transketolase. Thiamin pyrophosphate-derived transition states for transketolase and pyruvate dehydrogenase are not identical.

Thiamin thiazolone pyrophosphate (TTPP) has been reported to be an effective transition state analogue for the thiamin pyrophosphate-dependent partial reaction of pyruvate dehydrogenase (Gutowski, J. A., and Lienhard, G. E. (1976) J. Biol. Chem. 251, 2863-2866). The kinetics of the interaction of TTPP with transketolase are reported here. TTPP is a competitive inhibitor, with respect to thiamin pyrophosphate, of bakers' yeast transketolase but it is neither a tight binding inhibitor nor a slow binding inhibitor. TTPP decreases the kinetically observed negative cooperativity seen for thiamin pyrophosphate and also decreases the rate constant for the hysteretic activation of the enzyme by thiamin pyrophosphate. We conclude that thiamin thiazolone pyrophosphate is not an effective transition state analogue for the reaction catalyzed by bakers' yeast transketolase. This difference between transketolase and pyruvate dehydrogenase may be related to differences in the polarity of the active sites of the enzymes. It is conceivable that the active sites of the pyruvate decarboxylase subunit of pyruvate dehydrogenase is hydrophobic, by analogy with the known hydrophobicity of the active site of brewers' yeast pyruvate decarboxylase. This hydrophobicity would stabilize a transition state with no charge on the thiazole portion of the coenzyme, similar to the "uncharged" thiazole portion of TTPP. In contrast, the active site of bakers' yeast transketolase, which is known to contain charged amino acid side chains, should be less favorable for such an uncharged transition state. A charge-separated canonical form related to TTPP could be preferentially stabilized in the active site of transketolase.

Kinetics↗
PubMedDate not supplied

[Thiamine and pyruvate metabolism in tumor-bearing rats].

Correlation between pyruvate and lactate contents as well as between enzymes participating in turnover of the substrates (pyruvate- and lactate dehydrogenases, alanine aminotransferase, pyruvate kinase) were studied in rat liver tissue simultaneously with tumor growth and intensive thiaminotherapy. Thiamine, administered into rats carrying carcinosarcoma Woker-256 at a daily dose 12.5 mg/kg body weight, exhibited the normalizing effect on activity of enzymes studied, on quantitative content of LDH isoenzymes and on content of lactate in blood and of pyruvate in liver tissue. Possible effect of thiamine on pyruvate metabolism in tumoral impairment is discussed.

Animals↗
PubMed1983

Study on the role of SH-groups in the activity of muscle pyruvate dehydrogenase.

The kinetics of inactivation of the pyruvate dehydrogenase component of the pigeon breast muscle pyruvate dehydrogenase complex in the presence of 5,5'-dithiobis (2-nitrobenzoate) is biphasic. The rate constants for the fast and slow phases of the inactivation reaction are close to those for modification of two classes of SH-groups differing in their reactivities towards the inhibitor. The reaction order with respect to the inhibitor concentration suggests that the two distinct SH-groups are essential for the enzyme activity. Modification of these SH-groups results in inhibition of the overall activity of the pyruvate dehydrogenase complex and of the 2-hydroxyethyl thiamine pyrophosphate - acceptor oxidoreductase activity of its decarboxylating component. Thiamine pyrophosphate exerts a protective effect on the enzyme only at the slow phase of the enzyme inactivation and SH-modification. As a result of interaction between the holoenzyme and pyruvate (or apoenzyme and 2-hydroxyethyl thiamine pyrophosphate) the rate of the enzyme inactivation is increased. This is associated with masking of non-essential SH-groups and with an increase of the accessibility of two essential SH-groups to the inhibitor. The data obtained suggest the interrelationship between the essential SH-groups and the 2-hydroxyethyl thiamine pyrophosphate-acceptor oxidoreductase activity of pyruvate dehydrogenase.

2,6-Dichloroindophenol↗
PubMed1980

Bovine kidney pyruvate dehydrogenase complex. Limited proteolysis and molecular structure of the lipoate acetyltransferase component.

1. Bovine kidney pyruvate dehydrogenase multienzyme complex is inactivated by elastase in a similar manner as described earlier for papain. The core component, lipoate acetyltransferase, is cleaved by elastase into an active fragment (Mr 26000) and a fragment with apparent Mr of 45000 as analyzed by dodecylsulfate gel electrophoresis. Due to the fragmentation of the core, the enzyme complex is disassembled into its component enzymes which retain their complete enzymatic activities as assayed separately. 2. A different mechanism was found for the inactivation of pyruvate dehydrogenase complex with trypsin and some other proteases (chymotrypsin, clostripain). In these cases, the pyruvate dehydrogenase component is inactivated rapidly by limited proteolysis. More slowly, the enzyme complex is disassembled simultaneously with fragmentation of the lipoate acetyltransferase which again results in an active fragment of Mr 26000 and another fragment of apparent Mr 45000. Upon prolonged proteolysis, the latter fragment is cleaved further to give products of Mr 36000 or lower. 3. The enzyme-bound lipoyl residues of the pyruvate dehydrogenase complex have been labelled covalently by incubation with [2-14C]pyruvate. After treatment of this [14C]acetyl-enzyme with papain, elastase, or trypsin, radioactivity was associated exclusively with the 45000-Mr and 36000-Mr fragments but not with the active 26000-Mr fragment. 4. It is concluded that the bovine kidney lipoate acetyltransferase core is composed of 60 subunits each consisting of two dissimilar folding domains. One of these contains the intersubunit binding sites as well as the active center for transacylation whereas the other possesses the enzyme-bound lipoyl residues.

Acetyltransferases↗
PubMed1980

Detection of myocardial ischemia before infarction, based on accumulation of labeled pyruvate.

To determine whether ischemic, but not irreversibly injured myocardium, can be differentiated from normal tissue based on accumulation of labeled pyruvate, isolated hearts were perfused with buffer containing [14C]pyruvate under conditions of normal or low flow. Fifteen minutes after the hearts were exposed to labeled material, myocardial radioactivity was fourfold greater in ischemic compared to control hearts, due to accumulation of label in sequestered lactate produced from the pyruvate. Open-chest rabbits subjected to coronary occlusion exhibited a 1.73:1 ratio of radioactivity in ischemic compared with normal myocardium 15 min after systemic injection of [14C]pyruvate. The results obtained suggest that zones of myocardial ischemia should be detectable in vivo by positron tomography after systemic administration of [11C]pyruvate as well.

Animals↗
PubMed1980

Affinity labeling of the active site of yeast pyruvate kinase by 5'-p-fluorosulfonylbenzoyl adenosine.

Yeast pyruvate kinase is irreversibly inactivated by 1.1 mM 5'-p-fluorosulfonylbenzoyl adenosine at pH 8.6 with an initial rate constant of 0.019 min-1. A plot of kinact versus the 5'-p-fluorosulfonylbenzoyl adenosine concentration yields a hyperbolic curve indicative of binding of the analog prior to reaction. Marked protection is afforded by phosphoenolpyruvate + fructose 1,6-diphosphate + Mg2+ or MgATP suggesting that reaction occurs within the active site. When assayed at less than saturating phosphoenolpyruvate concentrations, the inactivation caused by the reagent in the absence of added ligands appears slower, and reaction in the presence of phosphoenolpyruvate, fructose 1,6-diphosphate, and Mg2+ produces an activation of the enzyme, the extent of which is dependent on the assay concentration of phosphoenolpyruvate. The rate constant for activation was observed to be 0.113 min-1. The activated enzyme exhibits both a lowered K0.5 and Hill coefficient compared to native pyruvate kinase. Subsequent addition of 5'-p-fluorosulfonylbenzoyl adenosine to activated pyruvate kinase in the absence of added ligands leads to inactivation with the rate constant independent of the assay concentration of phosphoenolpyruvate. Covalent reaction of pyruvate kinase with 5'-p-fluorosulfonylbenzoyl adenosine thus occurs at two distinct sites. In the presence of phosphoenolpyruvate, fructose 1,6-diphosphate, and Mg2+, incorporation of tritiated 5'-p-fluorosulfonylbenzoyl adenosine is linearly proportional to the extent of activation of the enzyme, with 4 mol of reagent bound/mol of tetrameric pyruvate kinase for maximally activated enzyme. In the absence of added ligands, approximately 4.5 mol of reagent are incorporated/mol of enzyme at 15 min of reaction, while 80% of the original activity remains. Subsequent incorporation is proportional to the extent of inactivation with 8 mol bound at 100% in activaton. In the presence of phosphoenolpyruvate, fructose 1,6-diphospate, and Mg2+, 3 tyrosines and 1 lysine residue, and in the absence of ligands, 6 tyrosines and 2 lysine residues are modified, suggesting that both amino acids are within the two nucleotide sites.

Adenosine↗
PubMed1981

Antiketogenic effect of gluconeogenic substrates. II. Effect of pyruvate.

The effect of pyruvate on the fate of free fatty acids in isolated hepatocytes from starved rats has been studied. 5 mM pyruvate inhibited ketone bodies production variously from endogenous substrates, acetate, octanoate and oleate. However, the incorporation of radioactivity into ketone bodies from acetate, octanoate or oleate was not affected by the presence of pyruvate. The oxidation of radioactive fatty acids to CO2 was increased by the presence of pyruvate. These results suggest that pyruvate inhibited ketogenesis by increasing the rate of the tricarboxylic acid cycle.

Animals↗
PubMed1978

Utilization of pyruvate, alanine and glutamate by isolated fat cells and their effects on glycerol metabolism.

To study the metabolic interactions of different substrates in adipose tissue in vitro, isolated fat cells from fed rats were incubated in medium containing either (U-14C)-pyruvate, L-(U-14C)-alanine, L-(U-14C)-glutamate or (1(-14)C)-glycerol, and supplemented or not with 5 mM glucose or with non-radioactive pyruvate, alanine or glutamate (2 or 10 mM). The utilization of pyruvate for CO2 or fatty acid formation was greater than that of alanine and glutamate, both in the absence and presence of glucose. Glucose enhanced the formation of fatty acids from all the labelled substrates, decreased the synthesis of glyceride glycerol from pyruvate and glycerol, and enhanced it from alanine and glutamate. Pyruvate and glutamate enhanced the utilization of glycerol by the adipocytes and these effects were significantly reduced in the presence of glucose. Thus, the metabolic fate of the glycerol taken up by the cells varied according to the nature of the available substrates.

Adipose Tissue↗
PubMed1980

Effect of phenformin on the metabolism of glucose, pyruvate and acetate in guinea-pig heart.

In the isolated perfused heart of the guinea-pig, phenformin could be shown to have a characteristic effect on cardiac performance and metabolism. In the working heart, phenformin (1 mmol1/1) decreased dp/dt and increased the end diatolic pressure; this reduced heart performance could not be explained by changes in the content of energy-rich compounds. Furthermore, phenformin increased lactate production in hearts perfused with glucose as substrate and inhibited the utilisation of pyruvate, but not of acetate. The activity of the purified pyruvate dehydrogenase complex was not influenced by phenformin, but the active form of the pyruvate dehydrogenase complex (PDHa) was diminished. This effect may be explained by an inhibition of the pyruvate dehydrogenase phosphatase. An inhibition of pyruvate dehydrogenase and, coincidently, of oxygen consumption might contribute to the development of lactic acidoses and, particularly, might be deleterious for the heart.

Adenine Nucleotides↗