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PubMed1987

Effects of chronic renal failure on the regulation of pyruvate kinase.

The effects of chronic renal failure on the enzyme activity of pyruvate kinase and the mRNA level of this enzyme were studied in 7 out of 8 nephrectomized rats. The mRNA level was measured by RNA-DNA dot blot hybridization, using cloned pyruvate kinase cDNA as hybridized probe. Neither the activity of M1-type pyruvate kinase nor the level of this enzyme in rat gastrocnemius muscle was affected by chronic renal failure, whereas L-type pyruvate kinase enzyme activity in uremic rat liver was lower than that in control at both fasted and refed states. The levels of L-type pyruvate kinase mRNA were not different between two groups at the fasted state. Induction of L-type pyruvate kinase mRNA after high carbohydrate diet refeeding was suppressed proportionally to the severity of chronic renal failure, which was expressed by the serum creatinine concentrations (r = -.876, P less than .005). These results indicate that the suppression of L-type pyruvate kinase activity in uremia was partly reflected by the decreased accumulation of this enzyme mRNA. There was a significantly negative correlation between L-type pyruvate kinase mRNA levels and plasma glucagon/insulin ratios (r = -.719, P less than .05). Hyperglucagonemia in uremia might play a major role in this suppression.

Animals↗
PubMed1992

Some kinetic properties of pyruvate kinase from Trypanosoma brucei.

We have studied the kinetics of the allosteric interactions of pyruvate kinase from Trypanosoma brucei. The kinetics for phosphoenolpyruvate depended strongly on the nature of the bivalent metal ions. Pyruvate kinase activated by Mg2+ had the highest catalytic activity, but also the highest S0.5 for phosphoenolpyruvate, while the opposite was true for pyruvate kinase activated by Mn2+. The reaction rates of Mg(2+)-pyruvate kinase and Mn(2+)-pyruvate kinase were clearly allosteric with respect to phosphoenolpyruvate, while the kinetics with Co(2+)-pyruvate kinase were hyperbolic. However, Co(2+)-pyruvate kinase was still sensitive to heterotropic activation. Trypanosomal pyruvate kinase is unique in that the best activator was fructose 2,6-bisphosphate. Ribulose 1,5-bisphosphate and 5-phosphorylribose 1-pyrophosphate were also strong heterotropic activators, which were much more effective than fructose 1,6-bisphosphate and glucose 1,6-bisphosphate. In the presence of the heterotropic activators, the sigmoidal kinetics with respect to phosphoenolpyruvate and the bivalent metal ions were modified as were the concentrations of phosphoenolpyruvate and the bivalent metal ions needed to attain the maximal activity. Maximal activities were not significantly changed with Mg2+ and Mn2+ as the activating metal ions. Moreover, with Co2+ and fructose 2,6-bisphosphate or ribulose 1,5-bisphosphate or 5-phosphorylribose 1-pyrophosphate, the maximal activity was significantly reduced. Ribulose 1,5-bisphosphate and 5-phosphorylribose 1-pyrophosphate resembled fructose 2,6-bisphosphate rather than fructose 1,6-bisphosphate and glucose 1,6-bisphosphate in their action in that the K0.5 values for the former 3 compounds increased when Mg2+ was replaced by Co2+, while the K0.5 for fructose 1,6-bisphosphate and glucose 1,6-bisphosphate increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Regulation↗
PubMed1993

Purification and characterization of pyruvate ferredoxin oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus.

Pyrococcus furiosus grows optimally at 100 degrees C by carbohydrate fermentation. It is thought to contain a novel tungsten-dependent, NAD(P)-independent glycolytic pathway in which one of the oxidation steps is catalyzed by a tungsten-containing aldehyde ferredoxin oxidoreductase. The enzyme that catalyzes the terminal oxidation step, pyruvate ferredoxin oxidoreductase (POR), has now been purified. POR has a molecular mass of 100 kDa and is comprised of three subunits (45, 31 and 24 kDa). It lacks tungsten but contains thiamine pyrophosphate (TPP) and two ferredoxin-type [4Fe-4S] clusters per molecule which, by EPR spectroscopy, can be differentiated by their relaxation properties. The enzyme requires CoASH but not TPP for pyruvate oxidation activity and will not use 2-oxoglutarate, phenyl pyruvate or indole pyruvate as substrates. POR is virtually inactive at 25 degrees C and shows a temperature optimum for pyruvate oxidation above 90 degrees C. The apparent Km values for pyruvate, CoASH and P. furiosus ferredoxin at 80 degrees C are 460, 100 and 70 microM, respectively. Carbon monoxide was a potent inhibitor of pyruvate oxidation (apparent Ki = 7 microM). The half-life of activity (t50%) in air at 25 degrees C was 15 min and the t50% value at 80 degrees C (under anaerobic conditions) was 23 min. Based on molecular comparisons with PORs from mesophilic organisms, it is proposed that P. furiosus POR may represent an ancestral form of a pyruvate-oxidizing enzyme.

Amino Acids↗
PubMed2006

Pyruvate protects mitochondria from oxidative stress in human neuroblastoma SK-N-SH cells.

Oxidative stress is implicated in neurodegenerative diseases including stroke, Alzheimer's disease and Parkinson's disease, and has been extensively studied as a potential target for therapeutic intervention. Pyruvate, a natural metabolic intermediate and energy substrate, exerts antioxidant effects in brain and other tissues susceptible to oxidative stress. We tested the protective effects of pyruvate on hydrogen peroxide (H(2)O(2)) toxicity in human neuroblastoma SK-N-SH cells and the mechanisms underlying its protection. Hydrogen peroxide insult resulted in 85% cell death, but co-treatment with pyruvate dose-dependently attenuated cell death. At concentrations of >or=1 mM, pyruvate totally blocked the cytotoxic effects of H(2)O(2). Pyruvate exerted its protective effects even when its administration was delayed up to 2 h after H(2)O(2) insult. As a scavenger of reactive oxygen species (ROS), pyruvate dose-dependently attenuated H(2)O(2)-induced ROS formation, assessed from 2,7-dichlorofluorescein diacetate fluorescence. Furthermore, pyruvate suppressed superoxide production by submitochondrial particles, and attenuated oxidative stress-induced collapse of the mitochondrial membrane potential. Collectively, these results suggest that pyruvate protects neuronal cells through its antioxidant actions on mitochondria.

Cell Death↗
PubMed2003

Effects of pyruvate administration on infarct volume and neurological deficits following permanent focal cerebral ischemia in rats.

Recent experimental evidences indicate that pyruvate, the final metabolite of glycolysis, has a remarkable protective effect against different types of brain injury. The purpose of this study was to assess the neuroprotective effect and the neurological outcome after pyruvate administration in a model of ischemic stroke induced by permanent middle cerebral artery occlusion (pMCAO) in rats. Three doses of pyruvate (250, 500 and 1000 mg/kg, i.p.) or vehicle were administered intraperitoneally 30 min after pMCAO. In other set of experiments, pyruvate was given either before, immediately after ischemia or in a long-term administration paradigm. Functional outcome, mortality and infarct volume were determined 24 h after stroke. Even when the lowest doses of pyruvate reduced mortality and neurological deficits, no concomitant reduction in infarct volume was observed. The highest dose of pyruvate increased cortical infarction by 27% when administered 30 min after pMCAO. In addition, when pyruvate was given before pMCAO, a significant increase in neurological deficits was noticed. Surprisingly, on the contrary of what was found in the case of transient global ischemia, present findings do not support a great neuroprotective role for pyruvate in permanent focal cerebral ischemia, suggesting two distinct mechanisms involved in the effects of this glycolytic metabolite in the ischemic brain.

Animals↗
PubMed2002

Potentiation of beta-adrenergic inotropic response by pyruvate in failing human myocardium.

BACKGROUND: Pyruvate has been shown to increase contractile function in isolated myocardium and to improve hemodynamics in patients with congestive heart failure. We tested the hypothesis that pyruvate potentiates the inotropic response to beta-adrenergic stimulation and to elevated extracellular calcium, since this may be of potential therapeutic value in the clinical setting of acute heart failure in order to circumvent deleterious effects on energy demand as can occur during catecholamine therapy. METHODS AND RESULTS: We investigated isometrically contracting isolated multicellular muscle preparations from terminal failing human hearts at 37 degrees C, pH 7.4, and a stimulation frequency of 1 Hz. At an extracellular calcium concentration of 1.25 mM, pyruvate (10 mM) alone increased developed force (F(dev)) from 9.0+/-2.3 to 21.1+/-4.3 mN/mm(2) (n=9, P<0.001) and isoproterenol (1 microM) alone increased F(dev) from 9.5+/-2.0 to 31.3+/-5.4 mN/mm(2) (P<0.001), whereas the combination of pyruvate and isoproterenol increased F(dev) over-proportionally from 9.0+/-2.3 to 47.4+/-6.4 mN/mm(2) (P<0.01). In a separate series we assessed the combination of pyruvate and calcium. Although F(dev) did not increase from 12 to 16 mM [Ca(2+)](o), 10 mM pyruvate further increased F(dev) from 25.8+/-5.0 to 30.6+/-4.7 mN/mm(2) (P<0.01). Rapid cooling contractures revealed that altered myofilament responsiveness and/or sarcoplasmic reticulum (SR) calcium load must underlie the positive inotropic effect of pyruvate. CONCLUSION: A combination of pyruvate and beta-adrenergic stimulation may be of therapeutic value in acute heart failure by reducing the concentrations of potential deleterious catecholamines that are currently necessary to maintain adequate tissue perfusion.

Adrenergic beta-Agonists↗
PubMed1999

Haemodynamic effects of intracoronary pyruvate in patients with congestive heart failure: an open study.

BACKGROUND: Pyruvate, as an intermediate in the Krebs cycle, is an important source of energy for myocardium and improves contractility of normal, hypoxic, and postischaemic animal myocardium. We investigated the effect of intracoronary pyruvate in patients with congestive heart failure. METHODS: Haemodynamic measurements were done in eight patients with dilated cardiomyopathy after two 15 min infusions of pyruvate into the left main coronary artery and after saline washout of pyruvate. FINDINGS: There were no significant differences between the two pyruvate concentrations. Application of pyruvate resulted in a 23% increase in cardiac index (p<0.05), a 38% increase in stroke-volume index (p<0.05), and a 36% decrease in pulmonary capillary wedge pressure (p<0.05). Heart rate decreased significantly by 11%. Mean aortic pressure and systemic vascular resistance did not change. Most of the effects of pyruvate were reversed 15 min after the infusion stopped. INTERPRETATION: Pyruvate has the profile of a favourable inotropic substance. Other modes of administration need to be studied.

Cardiac Catheterization↗
PubMed1994

Characterization of an ancestral type of pyruvate ferredoxin oxidoreductase from the hyperthermophilic bacterium, Thermotoga maritima.

The hyperthermophilic bacterium, Thermotoga maritima, is a strict anaerobe that grows up to 90 degrees C by carbohydrate fermentation. We report here on its pyruvate ferredoxin oxidoreductase (POR), the enzyme that catalyzes the oxidation of pyruvate to acetyl-CoA, the terminal oxidation step in the conversion of glucose to acetate. T. maritima POR was purified to electrophoretic homogeneity under strictly anaerobic conditions. It has a molecular weight of 113,000 and comprises four dissimilar subunits with M(r) values of approximately 43,000, 34,000, 23,000, and 13,000. It contains thiamine pyrophosphate (TPP) and at least two ferredoxin-type [4Fe-4S] clusters per molecule, as determined by iron analysis and EPR spectroscopy. CoASH was absolutely required for pyruvate oxidation activity, while the addition of TPP was stimulatory. The apparent Km values at 80 degrees C for pyruvate, CoASH, and TPP were 14.5, 0.34, and 0.043 mM, respectively, and the corresponding apparent Vm values ranged from 154 to 170 mumol of pyruvate oxidized/min/mg (units/mg). The apparent Km and Vm values for T. maritima ferredoxin, the proposed physiological electron carrier for POR, were 26 microM and 280 units/mg, respectively. POR did not use 2-oxoglutarate, phenyl pyruvate, or indolyl pyruvate as substrates. The enzyme was extremely thermostable: the temperature optimum for pyruvate oxidation was above 90 degrees C, and the time for a 50% loss of activity (t50%) at 80 degrees C (under anaerobic conditions) was 15 h. The enzyme was also very sensitive to inactivation by oxygen, with a t50% in air at 25 degrees C of 70 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1983

Activation of yeast pyruvate carboxylase: interactions between acyl coenzyme A compounds, aspartate, and substrates of the reaction.

Chicken liver pyruvate carboxylase has an absolute requirement for short-chain acyl coenzyme A (CoA), whereas the same enzyme from yeast has less stringent requirements. The yeast enzyme has now been studied in an effort to elucidate the mechanism by which acyl-CoA stimulates pyruvate carboxylase activity. Yeast pyruvate carboxylase has an apparent basal level of activity above which CoA and acyl-CoAs of 2-20 carbons activate; the concentration of acyl-CoA required for half-maximum activation (K0.5) decreases as the chain length of the acyl moiety increases to 16 carbons. Activation of yeast pyruvate carboxylase by acyl-CoA is brought about in part by increasing the affinity of pyruvate carboxylase for two substrates, bicarbonate and pyruvate. The affinity of pyruvate carboxylase for bicarbonate is also increased by potassium ions. The observation of only low levels of activity in the absence of acyl-CoA or potassium ion leads to the conclusion that the basal activity so frequently referred to is probably due to the presence of activating monovalent cations. Pyruvate carboxylase from yeast probably has an absolute requirement for monovalent cations or acyl-CoA with a combination of the two being required for optimum conditions for maximal activity. Stimulation by acyl-CoA and inhibition by aspartate are mutually antagonistic with each affecting the activation or inhibition constant and the degree of cooperativity brought about by the other. The enzyme from liver is unaffected by aspartate.

Acyl Coenzyme A↗
PubMed1986

Phosphorylation-dephosphorylation of pyruvate dehydrogenase from bakers' yeast.

The pyruvate dehydrogenase complex was purified to homogeneity from bakers' yeast (Saccharomyces cerevisiae). No pyruvate dehydrogenase kinase activity was detected at any stage of the purification. However, the purified pyruvate dehydrogenase complex was phosphorylated and inactivated with purified pyruvate dehydrogenase kinase from bovine kidney. The protein-bound radioactivity was localized in the pyruvate dehydrogenase alpha subunit. The phosphorylated, inactive pyruvate dehydrogenase complex was dephosphorylated and reactivated with purified pyruvate dehydrogenase phosphatase from bovine heart. Tryptic digestion of the 32P-labeled complex yielded a single phosphopeptide, which was purified to homogeneity. The sequence of the phosphopeptide was established to be Tyr-Gly-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Thr-Thr-Tyr-Arg. This sequence is very similar to the sequence of a tryptic phosphotetradecapeptide derived from the alpha subunit of bovine kidney and heart pyruvate dehydrogenase: Tyr-His-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Val-Ser-Tyr-Arg.

Adenosine Triphosphate↗
PubMed1990

Slow-binding inhibition of the Escherichia coli pyruvate dehydrogenase multienzyme complex by acetylphosphinate.

The pyruvate analogue acetylphosphinate (CH3-CO-PO2H2) inhibits the pyruvate dehydrogenase component (E1) of the Escherichia coli pyruvate dehydrogenase multienzyme complex in a time-dependent process with biphasic reaction kinetics. The formation of an initial, rapidly reversible enzyme-inhibitor complex (EI) with an apparent Ki of 0.12 +/- 0.025 microM is followed by the conversion to a tighter complex (EI) at a maximal rate of k3 = 0.87 +/- 0.34 min-1. The inhibition is reversible (dissociation rate constant k4 = 0.038 +/- 0.002 min-1), requires the presence of the cofactors thiamin pyrophosphate and Mg2+, and is competitive with regard to pyruvate. The microscopic rate constants give a value of 5 nM for the overall dissociation constant [Ki = [E] [I]/[( EI] + [EI]) = Kik4/(k3 + k4)] compared with values of 10 and 3.5 nM obtained by steady-state methods. Thus acetylphosphinate binds by 5 orders of magnitude more tightly to pyruvate dehydrogenase than does pyruvate (Km = 0.35 mM). Acetylphosphinate also affects the pyruvate dehydrogenase complex fluorescence when excited at 290 nm in a time-dependent manner with a maximal rate constant of 0.99 min-1, suggesting a conformational change in the enzyme complex as the slow step in conversion of EI to EI (k3). All these features taken together suggest that the interaction of the pyruvate dehydrogenase with acetylphosphinate involves the formation of a thiamin pyrophosphate-acetylphosphinate adduct that resembles the normal reaction intermediate, 2-(1-carboxy-1-hydroxyethyl)thiamin pyrophosphate (alpha-lactylthiamin pyrophosphate).

Escherichia coli↗
PubMed1976

Magnetic resonance studies of the proximity and spatial arrangement of propionyl coenzyme A and pyruvate on a biotin-metalloenzyme, transcarboxylase.

A spin-labeled ester of CoA, R-CoA (3-carboxy-2,2,5,5-tetramethyl-1-pyrolidinyl-1-oxy CoA thioester), has been shown by competition studies using electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) to bind specifically to the propionyl-CoA binding sites of transcarboxylase. Titrations indicate 0.7 +/- 0.2 binding site for R-CoA per enzyme-bound biotin with a dissociation constant of 0.33 +/- 0.12 mM. Propionyl-CoA binds to this site with a 1.3-fold lower disonable agreement with kinetically determined inhibitor constants of CoA and propionyl-CoA and propionyl-CoA (D. B. Northrop (1969), J. Biol. Chem. 244, 5808). The bit of this spin-label on 1/T1 of water protons. The formation of a ternary transcarboxylase-R-CoA-pyruvate complex is suggested by the failure of pyruvate to displace R-CoA from the tight site and is established by the paramagnetic effects of enzyme-bound R-CoA on the relaxation rates of the protons and 13C atoms of enzyme-bound pyruvate. From the paramagnetic effects of R-CoA on the relaxation rates of the methyl protons of pyruvate at 40.5 and 100 MHz, and on the 13C-enriched carbonyl and carboxyl carbon atoms of pyruvate at 25.1 MHz, a correlation time of 7 nsec and distances from the bound nitroxide radical to the methyl protons, the carbonyl, and carboxyl carbon atoms of bound pyruvate of 7.9 +/- 0.7, 10.3 +/- 0.8, and 12.1 +/- 0.9 A, respectively, are calculated. These distances establish the close proximity of the CoA ester and keto acid sites on transcarboxylase. Together with the previously determined distances from the enzyme-bound (Co(II) to the methyl protons and 2 carbon atoms of bound pyruvate and to 12 protons and 3 phosphorus atoms of bound propionyl-CoA, the present distances are used to derive a composite model of the bound substrates in the overall transcarboxylation reaction. In this model the distance from the methyl carbon of pyruvate and the methylene carbon of propionyl-CoA, between which the carboxyl transfer takes place is only approximately 7 A. Depending on the detailed mechanism of the carboxyl transfer, the distance through which the carboxybiotin must migrate is therefore between 0 and 7 A. Hence the major role of the 14-A arm of carboxybiotin is not to permit a large carboxyl migration but, rather to permit carboxybiotin to traverse the gap which occurs at the interface of three subunits and to insinuate itself between the CoA and keto acid sites.

Binding Sites↗
PubMed1976

Fluorescence energy transfer measurements between ligand binding sites of the pyruvate dehydrogenase multienzyme complex.

The interaction of the pyruvate dehydrogenase multienzyme complex from Escherichia coli with 8-anilino-1-naphthalenesulfonate (ANS), pyruvate, and acetyl-CoA has been investigated using equilibrium binding, steady-state fluorescence, and fluorescence lifetime measurements. The fluorescnece of ANS is greatly enhanced when bound to the enzyme complex and to the pyruvate dehydrogenase component of the complex. Approximately 22 molecules of ANS are bound to a molecule of the complex with a binding constant of 3.69 muM in 0.03 M potassium potassium phosphate (pH 7.0). Direct and competitive binding measurements indicate that about 42 pyruvate binding sites are present per mole of enzyme complex which has been stripped of thiamine diphosphate; the number of binding sites is reduced to 28,5 in the presence of a saturating concentration of thiochrome diphosphate, a thiamine diphosphate analogue. The dissociation constant for pyruvate to the enzyme complex in the presence of thiochrome diphosphate is 308 muM in 0.02 M potassium phosphate (pH 7.0). Pyruvate, thiochrome diphosphate, and acetyl-CoA all displace ANS from the enzyme complex. In the cases of pyruvate and thiochrome diphosphate, the concentration dependence of the displacements suggests the displacement is allosteric, while in the case of acetyl-CoA direct competition appears to be involved. GTP decreased the effect of acetyl-CoA to the enzyme complex indicate that 24-26 bound acetyl-CoA molecules per complex can be readily displaced by ANS, and the binding of acetyl-CoA to these sites displays positive cooperativity. Fluorescence energy transfer measurements between bound ANS on the pyruvate dehydrogenase enzyme and FAD on the dihydrolipoyl dehydrogenase enzyme indicate, assuming the emission and absorption dipoles are randomly oriented, that these two probes must be at least 58 A apart in the intact complex.

Anilino Naphthalenesulfonates↗
PubMed2007

A structural factor responsible for substrate recognition by Bacillus sp. GL1 xanthan lyase that acts specifically on pyruvated side chains of xanthan.

Xanthan is a bacterial heteropolysaccharide composed of pentasaccharide repeating units, i.e., a cellobiose as a backbone and a trisaccharide consisting of two mannoses and one glucuronic acid as a side chain. Nonreducing terminal mannose residues of xanthan side chains are partially pyruvated. Bacillus sp. GL1 xanthan lyase, a member of polysaccharide lyase family 8, acts specifically on pyruvated side chains of xanthan and yields pyruvated mannose through a beta-elimination reaction by using a single Tyr255 residue as base and acid catalysts. Here we show structural factors for substrate recognition by xanthan lyase through X-ray crystallographic and mutational analyses. The enzyme accommodates mannose and pyruvated mannose at the -1 subsite, although both inhibitor and dissociation constants of the two monosaccharides indicated that the affinity of pyruvated mannose for xanthan lyase is much higher than that of mannose. The high affinity of pyruvated mannose is probably due to the formation of additional hydrogen bonds between the carboxyl group of pyruvated mannose and amino acid residues of Tyr315 and Arg612. Site-directed mutagenesis of the two residues demonstrated that Arg612 is a key residue in recognizing pyruvated mannose. Arg612 is located in the protruding loop covering the substrate, suggesting that the loop functions as a lid that is responsible for the proper accommodation of the substrate at the active site.

Bacillus↗
PubMed2003

Cytoprotection of pyruvic acid and reduced beta-nicotinamide adenine dinucleotide against hydrogen peroxide toxicity in neuroblastoma cells.

Elevated production of hydrogen peroxide (H2O2) in the central nervous system has been implicated in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease, ischemic reperfusion, stroke, and Alzheimer's disease. Pyruvic acid has a critical role in energy metabolism and a capability to nonenzymatically decarboxylate H2O2 into H2O. This study examined the effects of glycolytic regulation of pyruvic acid on H2O2 toxicity in murine neuroblastoma cells. Glycolytic energy substrates including D-(+)-glucose, D-(-) fructose and the adenosine transport blocker dipyridamole, were not effective in providing protection against H2O2 toxicity, negating energy as a factor. On the other hand, pyruvic acid completely prevented H2O2 toxicity, restoring the loss of ATP and cell viability. H2O2 toxicity was also attenuated by D-fructose 1,6 diphosphate (FBP), phospho (enol) pyruvate (PEP), niacinamide, beta-nicotinamide adenine dinucleotide (beta-NAD+), and reduced form (beta-NADH). Both FBP and PEP exerted positive kinetic effects on pyruvate kinase (PK) activity. Interestingly, only pyruvic acid and beta-NADH exhibited powerful stoichiometric H2O2 antioxidant properties. Further, beta-NADH may exert positive effects on PK activity. Subsequent pyruvic acid accumulation can lead to the recycling of beta-NAD+ through lactate dehydrogenase and beta-NADH through glyceraldehyde-3-phosphate dehydrogenase. It was concluded from these studies that intracellular pyruvic acid and beta-NADH appear to act in concert through glycolysis, to enhance H2O2 intracellular antioxidant capacity in neuroblastoma cells. Future research will be required to examine whether similar effects are observed in primary neuronal culture or intact tissue.

Animals↗
PubMed1972

The effect of phenylpyruvate on pyruvate metabolism in rat brain.

1. The effect of phenylalanine and phenylpyruvate on the metabolism of pyruvate by isolated mitochondria from rat brain was investigated. 2. Phenylpyruvate inhibited the fixation of H(14)CO(3) (-) in the presence of pyruvate by intact rat brain mitochondria, whereas phenylalanine and other metabolites of this amino acid had no inhibitory effect on this process. 3. Pyruvate carboxylase activity in freeze-dried rat brain mitochondrial preparations was also inhibited only by phenylpyruvate, and a ;mixed type' inhibition was observed. 4. The K(m) for pyruvate of rat brain pyruvate carboxylase was about 0.2mm. 5. The concentration of phenylpyruvate required for a 50% inhibition of H(14)CO(3) (-) fixation by the intact mitochondria and of pyruvate carboxylase activity was dependent on the concentration of pyruvate used in the incubation medium. 6. The possible significance of inhibition of pyruvate carboxylase activity by phenylpyruvate in the brains of phenylketonuric patients is discussed.

Animals↗
PubMed1978

Stimulation of proinsulin biosynthesis and insulin release by pyruvate and lactate.

Increasing concentrations of pyruvate failed to stimulate proinsulin biosynthesis and insulin release in freshly isolated islets. Glycolytic flux (3H2O from [5-3H]glucose) decreased by 80-85%, but decarboxylation of [1(-14)C]pyruvate was unaffected in islets tested immediately after alloxan exposure. This strongly suggested that in freshly isolated islets, beta-cells, in relation to other islet cells, hardly contribute to the decarboxylation of pyruvate. Non-alloxan-treated cultured islets decarboxylated 2-2.5 times as much pyruvate as did alloxan-treated islets cultured for 15-18h. Thus the contribution of beta-cells to the metabolism of pyruvate after culturing markedly increased. Concomitantly beta-cells became responsive to pyruvate. At 20mM-pyruvate, release of prelabelled proinsulin and insulin and incorporation of [3H]leucine into proinsulin reached values approximately half of those obtained with 20mM-glucose. Lactate was as effective as pyruvate in inducing responses in cultured islets. The experiments indicate that a critical degree of substrate utilization is necessary for the generation of signals for insulin release and proinsulin biosynthesis.

Alloxan↗
PubMed1980

Biosynthesis of rat liver pyruvate kinase. Measurement of enzyme lifetime and the rate of synthesis at weaning.

Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of immunoprecipitates of liver cytosol with anti-(L-type pyruvate kinase) serum revealed proteins of mol.wt. 56 000 and 42 000 in addition to the heavy and light chains. The ratio of the 56 000 mol.wt. to the 42 000 mol.wt. protein increased under dietary conditions that resulted in an increase in the apparent specific activity of hepatic pyruvate kinase. The 42 000 mol.wt. protein was removed from immunoprecipitates if the liver cytosol was partially purified by pH precipitation and (NH4)2SO4 fractionation before addition of the antiserum. This technique may be used to analyse the formation of pure L-type pyruvate kinase in liver. By using H14CO3-labelling, the t1/2 of L-type pyruvate kinase was estimated as 75 +/- 1.7 h in post-weaned high-carbohydrate-diet-fed rats. Before weaning there was little immunoreactive pyruvate kinase in rat liver cytosol. Induction began between 6 and 24 h after weaning and reached a maximum value 120 h after weaning. When clearly enhanced total pyruvate kinase activity was first observed at 24 h post-weaning, the apparent specific activity of hepatic pyruvate kinase was considerably lower than the specific activity of the pure isolated enzyme. When the induction of L-type pyruvate kinase was monitored by the incorporation of L-[4,5-3H]leucine, the maximum rate of synthesis occurred 24--48 h after weaning. After this period synthesis declined, indicating a relatively slow turnover of the enzyme once the enzyme concentration was established in the liver.

Animals↗