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PubMed2005

Bench-to-bedside review: Amelioration of acute renal impairment using ethyl pyruvate.

Inflammation and oxidative stress cause renal impairment. Renal failure exacerbates the effect of oxidative stress on many organ systems. Antioxidants can prevent or treat renal failure in various experimental models and clinical situations. Pyruvate is an endogenous antioxidant with beneficial effects in animal models of oxidative stress. Because sodium pyruvate rapidly degrades in solution, a simple derivative of pyruvic acid, namely ethyl pyruvate, has been investigated as a therapeutic agent in preclinical studies. Ethyl pyruvate reduces organ system damage in ischaemia/reperfusion injury and haemorrhagic and endotoxic shock, at least in part through its antioxidant action. In addition, ethyl pyruvate appears to have direct beneficial effects on cytokine expression and proinflammatory gene regulation. The effect is long lasting and, importantly, even when it is administered after the onset of inflammation it can ameliorate organ damage and improve survival. Ethyl pyruvate is a widely used as a food additive and was shown to be safe in phase I clinical trials. We suggest ethyl pyruvate warrants further evaluation in the management of acute renal impairment.

Acute Kidney Injury↗
PubMed1979

The effects of season and temperature on D-lactate dehydrogenase, pyruvate kinase and arginine kinase in the foot of Helix pomatia L.

The effects of pH, season, environmental and experimental temperatures on the activities and kinetic parameters of D-lactate dehydrogenase, pyruvate kinase and arginine kinase from the foot of the pulmonate snail Helix pomatia were analyzed. Both in phosphate and Tris buffers D-lactate dehydrogenase was the enzyme with the most acid maximum, arginine kinase that with the most alkaline, whilst pyruvate kinase occupied an intermediate position. Pyruvate kinase activity, measured at 20 degrees C, was positively correlated with the environmental temperature at the moment of collecting the animal, whereas neither arginine kinase nor D-lactate dehydrogenase showed such a relationship. A seasonal study based on approximately 100 specimens established that arginine kinase activity remained the same throughout the year. Pyruvate kinase activity was slightly lower, and D-lactate dehydrogenase activity significantly higher, in winter than in summer animals. Snails subjected in spring to a short warm-up period before enzyme extraction showed extreme variability and some extraordinarily high values of pyruvate kinase activity, suggesting that either season or elevated temperature may have an immediate effect on the activity of this enzyme. Individual variability of all three enzymes ranges from 300 to 400%. The activities of pyruvate kinase and D-lactate dehydrogenase are strongly correlated in summer, forming a "constant-proportion-group", whereas in winter, with D-lactate dehydrogenase activity increasing and pyruvate kinase activity decreasing these two enzymes become "uncoupled". The Km value of pyruvate kinase is independent of experimental temperature between 10 and 25 degrees C, whereas that of D-lactate dehydrogenase and arginine kinase increases about three-fold within this range. Thus the temperature relationship of a single enzymic reaction cannot be used as an arguemnt for or against the occurrence of temperature compensation of whole animal metabolism. The possibility of modulation of enzyme activity by environmental temperature is discussed.

Acclimatization↗
PubMed2002

Effect of pyruvate on the function of stallion spermatozoa stored for up to 48 hours.

Stallion spermatozoa maintain high fertilizing capacity if cooled to 5 degrees C and inseminated within 24 h. However, if spermatozoa are stored for 48 h, fertilizing capacity declines. Therefore, multiple shipments of semen are often required to inseminate mares that remain in estrus for days. Therefore, experiments were designed to determine if adding antioxidants to stallion spermatozoa stored at 5 degrees C for 48 h could maintain motility and fertilizing ability. In the first experiment stallion spermatozoa were incubated in a skim milk (SM) or a skim milk-egg yolk medium in combination with 10 mM pyruvate, 5 mM xanthurenic acid separately or in combination for up to 48 h at 5 degrees C. Spermatozoa incubated in SM for 48 h exhibited higher percentages of motile sperm (57%) than did sperm incubated in skim milk-egg yolk (34%); antioxidant treatment had little effect. In the second experiment, spermatozoa were incubated in SM containing 0, 1, 2, or 5 mM pyruvate. After 24 h of incubation at 5 degrees C, sperm incubated with 1, 2, or 5 mM pyruvate exhibited higher percentages of progressively motile spermatozoa (45%) than control exhibited (26%; P < 0.05). After 48 h, percentages of progressively motile spermatozoa were similar (27, 19, and 30 vs 14, respectively; P > 0.05). However, when incubated at 5 degrees C for 48 h and then incubated an additional 4 h at 25 degrees C, samples containing pyruvate exhibited higher percentages of motile (63 to 80%) and progressively motile (36 to 42%) sperm than did sperm in SM alone (28 and 5%, respectively; P < 0.05). The third experiment attempted to determine the optimal pyruvate concentration to maintain spermatozoal motility. Spermatozoa incubated with 0, 2, 3.5, or 5 mM pyruvate for 48 h at 5 degrees C and then an additional 4 h at 25 degrees C, exhibited similar percentages of progressively motile cells (31, 35, and 28%, respectively) that were higher than control (11%, P < 0.05). The last experiment evaluated the fertilizing potential of cooled spermatozoa. Embryos were recovered from 35, 20, and 30% of mares inseminated with spermatozoa that had been incubated at 5 degrees C, for 24 h in SM, or for 48 h in SM or SM + 2 mM pyruvate, respectively (P > 0.05). These studies indicate that 2 mM pyruvate in SM was beneficial in maintaining spermatozoal motility in 48 h-stored sperm and, although not significant, seemed to help maintain the fertility of 48 h-cooled spermatozoa.

Animals↗
PubMed1975

Investigation into pyruvate kinases from Escherichia coli K-12 grown under aerobic and anaerobic conditions.

Two forms of anaerobic Escherichia coli K-12 pyruvate kinase (EC 2.7.1.40) were separated by ammonium sulphate fractionations. Pyruvate kinases I is allosteric and pyruvate kinase II is non-allosteric to phosphoenolpyruvate. The addition of 1 mM FDP reversed the allostery to normal Michaelis-Menten kinetics. AMP had no effect, whereas 8 mM ATP completely inhibited the enzyme. The enzyme showed normal kinetics with ADP as substrate. Mg2+ and Mn2+ stimulated whereas Cu2+ severely inhibited the enzyme, which could be reversed by the addition of 1 mM FDP. Citrate, alpha-ketoglutarate, succinate, fumarate and alanine inhibited the enzyme, whereas phenylalanine had no effect. The allosteric pyruvate kinase from aerobic cultures was not only activated by FDP, but also by AMP. FDP changed Km and Vmax, whereas AMP influenced only the Km. During aerobic-anaerobic transition, pyruvate kinase synthesis increases and reaches a maximum under anaerobic conditions. The degree of FDP activation remains constant, but AMP activation is lost during transition. Aerobic cultures of E. coli K-12 grown on gluconeogenic substrates exhibited pyruvate kinase II activity (non-allosteric), which was stimulated by FDP and by AMP. It has been suggested that E. coli may have two types of pyruvate kinase II depending on the substrate and two types of pyruvate kinase I depending on oxygen tension in the medium.

Adenine Nucleotides↗
PubMed1975

The hormonal control of gluconeogenesis by regulation of mitochondrial pyruvate carboxylation in isolated rat liver cells.

The possibility that hormones control hepatic gluconeogenesis via the regulation of the rate of mitochondrial pyruvate carboxylation was investigated with the use of suspensions of liver cells isolated from fasted rats. The mitochondria prepared from liver cells were judged in good condition as they exhibited satisfactory phosphorus-oxygen and respiratory control ratios and transported Ca2+ and K+ ions in an energy-dependent manner. Addition of glucagon, epinephrine, or cyclic adenosine 3':5'-monophosphate to liver cells caused a 50 to 80% increase in the rate of glucose synthesis from lactate. When mitochondria were isolated from the cells after treatment with these agonists, they displayed 2- to 3-fold increases in the rate of pyruvate carboxylation, pyruvate decarboxylation, and pyruvate uptake. These mitochondrial changes are similar to those obtained in hepatic mitochondria prepared from intact, hormone-treated rats. The mitochondrial responses were specific for agents that stimulated gluconeogenesis; no response occurred with 5'-AMP or cyclic adenosine 2':3'-monophosphate. In the cell suspensions, the dose response curves for the activation of mitochondrial pyruvate metabolism and for increased glucose synthesis from L-lactate were coincident with four different agonists. The mitochondrial changes resulting from stimulation with glucagon developed in 1 to 2 min after the rise in cyclic adenosine 3':5'-monophosphate and occurred at least as early as the increase in the rate of gluconeogenesis. When the intracellular level of cyclic adenosine 3':5'-monophosphate returned to basal values, the rates of mitochondrial pyruvate carboxylation and glucose synthesis also declined to control levels. It is concluded that the rate of mitochondrial pyruvate metabolisms can be increased by hormones and cyclic nucleotides and that control of mitochondrial pyruvate carboxylation is an important regulatory site of hepatic gluconeogenesis.

Animals↗
PubMed1976

Inhibition of gluconeogenesis and lactate formation from pyruvate by N6, O2'-dibutyryl adenosine 3':5'-monophosphate.

N6,O2-Dibutyryl adenosine 3':5'-monophosphate (Bt2cAMP) inhibits gluconeogenesis and lactate formation but increases ketogenesis by isolated liver cells incubated with high concentrations of pyruvate. The inhibitory effects can not be explained on the basis of an inhibition of the pyruvate dehydrogenase complex nor by a change in the NAD+ oxidation-reduction potential of the mitochondrial compartment. Both oleate and 3-hydroxybutyrate substantially increase the rates of gluconeogenesis and lactate formation from pyruvate but do not overcome the inhibition caused by Bt2cAMP. A decreased effectiveness of pyruvate kinase is proposed to account for the inhibition of both gluconeogenesis and lactate formation by Bt2cAMP. This enzyme catalyzes a step required in the transfer of reducing equivalents from the mitochondrial compartment to the cytoplasm and participates in the formation of glucose and lactate from pyruvate by the overall reaction: 2 pyruvate- + 2 NADHmito + 4 ATP4- + 4 H2O leads to 1/2 glucose + lactate- + 2 NAD+ mito + 4 ADP3- + 4 HPO4(2)- + H+. Inhibition of pyruvate kinase promotes gluconeogenesis with most substrates but inhibits gluconeogenesis from pyruvate for want of cytoplasmic reducing equivalents.

Animals↗
PubMed1986

Transcriptional and post-transcriptional regulation of L-type pyruvate kinase gene expression in rat liver.

The effects of starvation, refeeding a diet high in carbohydrate, administration of glucagon and cyclic AMP, thyroidectomy, and adrenalectomy on transcription of the gene for liver L-type pyruvate kinase and on the accumulation of cytoplasmic mRNA for L-type pyruvate kinase were investigated in rat. Transcription of the gene was undetectable in either fasted or protein-fed rats. Refeeding fasted rats a carbohydrate-rich diet stimulated an increase in L-type pyruvate kinase mRNA, preceded by an increase in the gene transcription. Transcription was maximal at 12 h of refeeding, decreasing to 10% of maximum at 72 h. The level of L-type pyruvate kinase mRNA remained constant at 50% of maximum for at least 120 h. Neither thyroidectomy nor adrenalectomy affected gene transcription in fasted rats refed the carbohydrate-rich diet, despite a decrease in mRNA abundance to 40 and 20%, respectively, of controls fed a normal diet. Glucagon or cyclic AMP totally blocked the increase in transcription of the L-type pyruvate kinase gene caused by feeding a carbohydrate-rich diet to previously fasted rats. Nevertheless, the level of L-type pyruvate kinase mRNA remained high for 3 h after glucagon administration. After 3 h, the mRNA decreased rapidly with a half-life less than 1 h. Thus, expression of the gene for L-type pyruvate kinase is regulated at both transcriptional and post-transcriptional levels. The transcription is regulated by two major effectors, one positive, namely carbohydrates, and one negative, namely glucagon (via cyclic AMP). Both agents probably act at the level of the mRNA stability as well. Glucocorticoids and thyroid hormones do not regulate transcription of the gene for L-type pyruvate kinase but do appear to be required for a normal accumulation of the transcripts in the cytoplasm.

Adrenalectomy↗
PubMed1971

Pathway of carbon flow during fatty acid synthesis from lactate and pyruvate in rat adipose tissue.

The metabolism of pyruvate and lactate by rat adipose tissue was studied. Pyruvate and lactate conversion to fatty acids is strongly concentration-dependent. Lactate can be used to an appreciable extent only by adipose tissue from fasted-refed rats. A number of compounds, including glucose, pyruvate, aspartate, propionate, and butyrate, stimulated lactate conversion to fatty acids. Based on studies of incorporation of lactate-2-(3)H and lactate-2-(14)C into fatty acids it was suggested that the transhydrogenation sequence of the "citrate-malate cycle"(1) was not providing all of the NADPH required for fatty acid synthesis from lactate. An alternative pathway for NADPH formation involving the conversion of isocitrate to alpha-ketoglutarate via cytosolic isocitrate dehydrogenase was proposed. Indirect support for this proposal was provided by the rapid labeling of glutamate from lactate-2-(14)C by adipose tissue incubated in vitro, as well as the demonstration that glutamate can be readily metabolized by adipose tissue via reactions localized largely in the cytosol. Furthermore, isolated adipose tissue mitochondria convert alpha-ketoglutarate to malate, or in the presence of added pyruvate, to citrate. Glutamate itself can not be metabolized by these mitochondria, a finding in keeping with the demonstration of negligible levels of NAD-glutamate dehydrogenase activity in adipose tissue mitochondria. Pyruvate stimulated alpha-ketoglutarate and malate conversion to citrate and reduced their oxidation to CO(2). It is proposed that under conditions of excess generation of NADH malate may act as a shuttle carrying reducing equivalents across the mitochondrial membrane. Malate at low concentrations increased pyruvate conversion $$Word$$ citrate and markedly decreased the formation of CO(2) by isolated adipose tissue mitochondria. Malate also stimulated citrate and isocitrate metabolism by these mitochondria, an effect that could be blocked by 2-n-butylmalonate. This potentially important role of malate in the regulation of carbon flow during lipogenesis is underlined by the observation that 2-n-butylmalonate inhibited fatty acid synthesis from pyruvate, but not from glucose and acetate, and decreased the stimulatory effect of pyruvate on acetate conversion to fatty acids.

Acetates↗
PubMed1984

Cooperativity in highly aggregated enzyme systems. A slow transition model for the pyruvate dehydrogenase complex from Escherichia coli.

Three models are compared describing cooperative phenomena in enzymatic reactions in order to explain sigmoidal saturation curves found with the pyruvate dehydrogenase complex from Escherichia coli: the concerted model, the sequential model, and the slow transition model. Both the concerted and the sequential model were considered especially with regard to the increasing number of identical interaction subunits (protomers) in order to get close to the situation found with the pyruvate dehydrogenase complex which consists of 24 protomers. Applying the sequential model to a great number of protomers results in a weak increase of the Hill coefficient, while, in addition to this effect, the concerted model drastically shifts the sigmoidal range of the saturation function to very low ligand concentrations. Such shift is seen with saturation curves of pyruvate and thiamine disphosphate with the pyruvate dehydrogenase complex and a good fit with theoretical curves derived from the concerted model is obtained. However, subcomplexes with a reduced number of protomers exhibited no change in saturation behavior, thus providing evidence against concerted conformational changes of all subunits of the enzyme complex. A scheme for the initial reaction of the pyruvate dehydrogenase complex based on slow transitions is presented and a rate equation has been derived. Ordered binding of thiamine diphosphate and pyruvate and a ligand-induced slow transition between a less active and a fully active enzyme form has been assumed. The curves simulated with this model are in agreement with all essential kinetic data, which are observed with the pyruvate dehydrogenase complex: the atypical shape of the saturation curves of pyruvate and thiamine diphosphate, the respective Hill coefficients and Michaelis constants, the hyperbolic binding behavior of thiamine diphosphate, and the inhibition pattern found for acetyl coenzyme A.

Escherichia coli↗
PubMed1984

Molecular cloning of a cDNA for human pyruvate carboxylase. Structural relationship to other biotin-containing carboxylases and regulation of mRNA content in differentiating preadipocytes.

An oligonucleotide probe specific for the amino acid sequence at the biotin site in pyruvate carboxylase was used to screen a human liver cDNA library. Nine cDNA clones were isolated and three proved to be pyruvate carboxylase clones based on nucleotide sequencing and Northern blotting. The biotin site amino acid sequence of human pyruvate carboxylase agreed perfectly with that of the sheep enzyme in 14 consecutive positions. The highly conserved amino acid sequence, Ala-Met-Lys-Met, found at the biotin site in most biotin-containing carboxylases was also present in human pyruvate carboxylase. The termination codon was located 35 residues 3' to the lysine residue at which the biotin is attached. Therefore, the biotin cofactor is covalently linked near the carboxyl-terminal end of the carboxylase protein. These data are consistent with that observed for other biotin-containing carboxylases and strongly suggests that the genes encoding the biotin-containing carboxylases may have evolved from a common ancestral gene. Northern blotting of mRNA isolated from human, baboon, and rat liver demonstrated that the pyruvate carboxylase mRNA was 4.2 kilobase pairs in length in all species examined. Southern blot analysis of genomic DNA isolated from human-Chinese hamster somatic cell hybrids localized the pyruvate carboxylase gene on the long arm of human chromosome 11. The human cDNA was also used to quantitate pyruvate carboxylase mRNA levels in a differentiating mouse preadipocyte cell line. These data demonstrated that pyruvate carboxylase mRNA content increased 23-fold in 7 days after the onset of differentiation.

Adipose Tissue↗
PubMed1983

Metabolic alterations in trauma: lactate and pyruvate levels after aortic surgery.

Arterial lactate and pyruvate concentrations were measured in five patients undergoing abdominal aneurysmectomy. In all cases, a bimodal rise and fall in both lactate and pyruvate levels were observed. Hemodynamic data indicated that cardiac output as well as oxygen delivery and consumption were constant throughout this period. The first lactate peak which was initially related to the cross-clamping of the aorta, but which continued for a period of 5 h after removal of the clamp, was characterized by elevated lactate to pyruvate ratios indicating an anaerobiosis. A transient hyperglycemia was also present during this lactate peak. Ten to 15 h later, when lactate and pyruvate levels were returning to normal levels, a second peak occurred in both lactate and pyruvate lasting 10 h in all five patients. A relatively large increase in pyruvate levels also occurred during this second peak so that the lactate to pyruvate ratios remained normal. This suggests that the second peak was characterized by aerobic metabolism and may have been due to a defect in pyruvate metabolism.

Aorta, Abdominal↗
PubMed1993

The pyruvate kinase gene as a model for studies of glucose-dependent regulation of gene expression in the endocrine pancreatic beta-cell type.

The insulinoma beta-cell line INS-1 expresses the L-type pyruvate kinase gene at high level and responds to a rise in extracellular glucose by strong induction of gene expression. Following the addition of glucose to the culture medium in the 3.5-33 mM concentration range, the cellular level of L-type pyruvate kinase mRNA increases within 2 h and reaches a maximum 15-fold above basal in 8-12 h. By run-on nuclear assay, the relative transcription rate of the pyruvate kinase gene is shown to increase 4-fold at maximal stimulation, suggesting that both transcriptional and post-transcriptional effects contribute to mRNA accumulation. The glucose effect is totally suppressed by the hexokinase inhibitor mannoheptulose, indicating a requirement for glucose phosphorylation. The mRNA induction is not inhibited in glutamine-free culture medium or by azaserine, suggesting that the hexosamine biosynthetic pathway is not involved. Moreover, metabolism along the glycolytic pathway does not appear to be an absolute requisite, since 2-deoxyglucose partly mimics the inductive effect of glucose. The glucose effect on the pyruvate kinase gene is reversibly antagonized by agents increasing intracellular cAMP. In addition, the effect is highly specific to the pyruvate kinase gene. Neither proinsulin I mRNA nor glucokinase mRNA are increased in glucose-stimulated INS-1 cells. Short term transfection with CAT plasmids driven by the pyruvate kinase L promoter reveals specific glucose-inducible reporter activity with the 183-base pair promoter region upstream of the cap site. Within this region, the previously described L4 cis-acting element is crucial for glucose responsiveness, as demonstrated by the fact that a plasmid with a mutation in this element does not elicit glucose-inducible CAT activity. Induction of L-type pyruvate kinase mRNA occurs in the islets of rats subjected to fasting and carbohydrate refeeding. In conclusion, the L-type pyruvate kinase gene provides an interesting model of glucose-regulated gene in the endocrine beta-cell type.

Animals↗
PubMed1993

Glucose catabolism in African trypanosomes. Evidence that the terminal step is catalyzed by a pyruvate transporter capable of facilitating uptake of toxic analogs.

The protozoan parasite Trypanosoma brucei derives its metabolic energy exclusively from a unique type of glycolysis in which pyruvate derived from glucose catabolism is released into the host bloodstream. In this study, this terminal metabolic step has been examined in detail. Pyruvate release from trypanosomal cells supplied with glucose is very rapid, proceeding with an apparent Vmax of 214 nmol x min-1 x mg-1. Counterflow experiments with [14C]pyruvate demonstrate that this metabolic end product can be taken up by actively metabolizing cells consistent with the presence of a plasma membrane transporter. The findings that [14C] acetate exhibits a much lower capacity for cell entry and that the structural analog alpha-cyano-3-hydroxycinnamic acid inhibits pyruvate release provide additional support for the presence of a pyruvate transporter. The substrate analog and alkylating agent 3-bromopyruvate inhibits completely both cell motility and pyruvate release. Surprisingly, however, it is a poor inhibitor of pyruvate transport per se. Rather, its preferential site of action and that of iodoacetic acid were identified by radiolabeling studies and microsequence analysis as glyceraldehyde-3-phosphate dehydrogenase. In extending these studies, 3-bromopyruvate was found to be over 20 times less effective in inhibiting glyceraldehyde-3-phosphate dehydrogenase in intact erythrocytes than in trypanosomal cells. However, in sonicated preparations from both cell types, the enzyme exhibits nearly identical sensitivities to inhibition by 3-bromopyruvate. Experiments reported here provide the first direct evidence that pyruvate release in African trypanosomes is catalyzed by a specific transport system and implicate this transporter as a vehicle for delivering toxic alkylating agents into trypanosomal cells.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1982

Determination of pyruvate dehydrogenase in cultured human fibroblasts and amniotic fluid cells.

A new assay method for pyruvate dehydrogenase in platelets [1] using in situ generation of [1-14C]pyruvate from [1-14C]lactate has been further developed for the use in cultured human fibroblasts and amniotic fluid cells. A very low blank rate was found compared to the method using [1-14C]pyruvate as substrate and an activity of 20 times the blank was obtained in normal fibroblasts and amniotic fluid cells. The pyruvate dehydrogenase was linear with time and protein concentration. The activity was greatly decreased by addition of non-radioactive pyruvate to the assay mixture and by preincubation with ATP. The cofactor requirement was similar to pyruvate dehydrogenase from other sources. Normal activities in cultured human fibroblasts and amniotic fluid cells were 13.9 pkat/mg protein (n = 15) and 21.7 pkat/mg protein (n = 10), respectively. The method was found to be very reliable and makes the diagnosis of pyruvate dehydrogenase deficiency possible in easily accessible tissue such as cultured fibroblasts. Prenatal diagnosis may also be a possibility.

Adenosine Triphosphate↗
PubMed1990

Pyruvate sparing by butyrate and propionate in proliferating colonic epithelium.

1. The effects of fasting and fasting followed by refeeding on the relative activities of the pyruvate dehydrogenase (PDH) complex and the tricarboxylic acid (TCA) cycle in isolated rat colonocytes were estimated by the rate of production of 14CO2 from [1-14C]pyruvate and [3-14C]pyruvate, respectively. 2. Decarboxylation of pyruvate by the PDH complex exceeded that by the TCA cycle in both fasted and fasted/refed colonocytes, was higher in distal than in proximal colon, and was stimulated by refeeding following a fast. 3. Oxidation of pyruvate by both the PDH complex and the TCA cycle was inhibited by butyrate. 4. Propionate alone had no effect, but synergized with butyrate to further reduce pyruvate decarboxylation by the TCA cycle. 5. Preferential utilization of butyrate by proliferating colonic epithelial cells is postulated to maximize the energy yield and spare pyruvate and its precursors for alternative synthetic roles necessary for active cell division.

Animals↗
PubMed1981

Metabolic consequences of pyruvate kinase inhibition by oxalate in intact rat hepatocytes.

The effects of oxalate on glycolysis and glucose production from trioses were studied in hepatocytes isolated from fed and fasted rats. 1--In cells from fed rats oxalate inhibited glycolysis at the pyruvate kinase step, as shown by an increased phosphoenolpyruvate concentration, a decreased lactate and pyruvate production and a reduction of the glycolytic flux estimated by the rate of detritiation of [6-3H] glucose. The plot of 1/lactate production versus oxalate concentration showed that pyruvate kinase is a limiting step of glycolysis and allowed to determine the apparent inhibition constant for oxalate: about 3035 microM which is near the physiological concentration of blood oxalate. Under conditions where both pyruvate kinase and glycolytic flux are inhibited, oxalate had no effect on the synthesis of [14C] glucose from [14C] triose. 2--In hepatocytes prepared from fasted rats and incubated with lactate and pyruvate, oxalate decreased gluconeogenesis. In cells isolated from fasted rats and incubated with dihydroxyacetone, oxalate decreased lactate and pyruvate production whereas glucose synthesis remained unchanged. It is concluded that the inhibition of pyruvate kinase cannot by itself increase the gluconeogenic flux from triose.

Animals↗
PubMed1994

Kinetic evidence for separate site catalysis by pyruvate phosphate dikinase.

Pyruvate phosphate dikinase from Clostridium symbiosum catalyzes the interconversion of adenosine-5'-triphosphate (ATP), orthophosphate (Pi), and pyruvate with adenosine 5'-monophosphate (AMP), inorganic pyrophosphate (PPi), and phosphoenolpyruvate (PEP) using a bi (ATP, Pi) bi (AMP, PPi) uni (pyruvate) uni (PEP) kinetic mechanism and pyrophosphorylenzyme (E-PP) and phosphorylenzyme (E-P) covalent intermediates. The present studies were carried out to determine whether or not the site of catalysis of the E + ATP + P1 E-P + AMP + PPi partial reaction overlaps with that of the E-P + pyruvate E + PEP partial reaction. Single-turnover experiments were carried out to test the effect of binding site occupancy on catalysis at a second site. Saturation of the enzyme with adenyl imidodiphosphate (AMPPNP) inhibited [32P]E-P formation from [beta-32P]ATP and Pi but did not significantly inhibit [32P]E-P formation from [32P]PEP. Likewise, saturation of E-P with AMP did not significantly inhibit [14C]PEP formation from [14C]pyruvate, suggesting separate, largely independent ATP/AMP vs pyruvate/PEP sites. Movement of the phosphorylhistidine residue between reaction sites was probed by testing oxalate as an inhibitor of phosphoryl transfer from E-P to [14C]pyruvate or to [14C]AMP. Both phosphoryl transfers were inhibited. These results were interpreted as evidence for the requirement for phosphorylhistidine release from the pyruvate site prior to participation in catalysis at the nucleotide site.

Adenosine Monophosphate↗
PubMed2001

Regulation of pyruvate dehydrogenase activity through phosphorylation at multiple sites.

The enzymic activity of the mammalian pyruvate dehydrogenase complex is regulated by the phosphorylation of three serine residues (sites 1, 2 and 3) located on the E1 component of the complex. Here we report that the four isoenzymes of protein kinase responsible for the phosphorylation and inactivation of pyruvate dehydrogenase (PDK1, PDK2, PDK3 and PDK4) differ in their abilities to phosphorylate the enzyme. PDK1 can phosphorylate all three sites, whereas PDK2, PDK3 and PDK4 each phosphorylate only site 1 and site 2. Although PDK2 phosphorylates site 1 and 2, it incorporates less phosphate in site 2 than PDK3 or PDK4. As a result, the amount of phosphate incorporated by each isoenzyme decreases in the order PDK1>PDK3>or=PDK4>PDK2. Significantly, binding of the coenzyme thiamin pyrophosphate to pyruvate dehydrogenase alters the rates and stoichiometries of phosphorylation of the individual sites. First, the rate of phosphorylation of site 1 by all isoenzymes of kinase is decreased. Secondly, thiamin pyrophosphate markedly decreases the amount of phosphate that PDK1 incorporates in sites 2 and 3 and that PDK2 incorporates in site 2. In contrast, the coenzyme does not significantly affect the total amount of phosphate incorporated in site 2 by PDK3 and PDK4, but instead decreases the rate of phosphorylation of this site. Furthermore, pyruvate dehydrogenase complex phosphorylated by the individual isoenzymes of kinase is reactivated at different rates by pyruvate dehydrogenase phosphatase. Both isoenzymes of phosphatase (PDP1 and PDP2) readily reactivate the complex phosphorylated by PDK2. When pyruvate dehydrogenase is phosphorylated by other isoenzymes, the rates of reactivation decrease in the order PDK4>or=PDK3>PDK1. Taken together, results reported here strongly suggest that the major determinants of the activity state of pyruvate dehydrogenase in mammalian tissues include the phosphorylation site specificity of isoenzymes of kinase in addition to the absolute amounts of kinase and phosphatase protein expressed in mitochondria.

Adenosine Triphosphate↗