Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.4.1.1 (pyruvate carboxylase)
1,516 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Pyruvate carboxylase activity was investigated in cultured fibroblasts from a patient shown to have hepatic pyruvate carboxylase deficiency. Under standard conditions, the activity in fibroblasts was 50% of controls (p less than 0.001). Kinetic investigations of the enzyme showed abnormal protein linearity with low activity at low protein concentration. Mixture of homogenates from the patient and a control revealed no endogenous inhibitor. Temperature stability of the mutant enzyme was similar to controls. Apparent kinetic constants for the substrates bicarbonate, ATP and pyruvate were in the patient 2.6 mmol/l, 0.08 mmol/l and 0.10 mmol/l compared to 2.1 mmol/l, 0.13 mmol/l and 0.22 mmol/l in controls, respectively. The 50% inhibitory concentration of oxaloacetate was 0.5 mmol/l in controls. However, no inhibitory effect of oxaloacetate was found for pyruvate carboxylase in fibroblasts from the patient. With acetyl-CoA, the apparent activation constant was 0.21 mmol/l in controls and 0.10 mmol/l in the patient, while the Hill coefficients were similar. These results may be explained by a mutation primarily affecting the transcarboxylation site of pyruvate carboxylase from the patient.
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PMID:A mutation of pyruvate carboxylase in fibroblasts from a patient with severe, chronic lactic acidaemia. 688 8

The potential activity of pyruvate carboxylase in lamprey liver is the same as in mammals. However, at certain stages of the life cycle this reaction does not take place because of ATP deficiency in mitochondria. Energy charge potential of liver cells ranges from 0.76 to 0.11 throughout a year. Heat adaptation of lampreys leads to a rapid increase of the ATP level and of the NAD+/NADH ratio in liver. The intensity of gluconeogenesis and glycogen levels are also enhanced. Cold reacclimation reverses the effect. A scheme accounting for the temperature changes in energy status of hepatocytes has been proposed.
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PMID:Switch on and switch off phenomenon of liver gluconeogenic function in lamprey (Lampetra fluviatilis L.) under the influence of season and temperature. 688 6

1. We have examined effects, on gluconeogenesis from lactate, of altering energy metabolism in two ways: (a) by primarily lowering cytosolic ATP levels with the use of atractyloside or 2,5 anhydromannose; and (b) by decreasing mitochondrial energy generation with the use of the classical uncoupler, dinitrophenol. 2. Agents which lower cytosolic ATP inhibit gluconeogenesis and increase pyruvate kinase flux (PK) correspondingly, while pyruvate carboxylase and P-enolpyruvate carboxykinase fluxes are unchanged, at least until gluconeogenesis is inhibited by more than 50%. 3. Dinitrophenol, on the other hand, although it also induces a (smaller) increase in PK, primarily decreases gluconeogenesis by an effect on a mitochondrial step in the gluconeogenic pathway. 4. Low concentrations of dinitrophenol increase Krebs cycle oxidation by at least 50% before significant inhibition of gluconeogenesis from lactate occurs.
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PMID:Effects of alterations in energy supply on gluconeogenesis from L-lactate. 711 74

Pyruvate carboxylase of the facultative methylotroph Pseudomonas oleovorans was purified 40-fold by ammonium sulfate fractionation, gel filtration on Ultrogel AcA 34, ion-exchange chromatography on DEAE-Biogel A and concentration on DEAE-Sepharose CL-6B. The enzyme exerts its maximal activity in the presence of Mg2+ (pH 7.5, 40 degrees), is unstable and completely inactivated within 6 hrs at 25 degrees. In the presence of Mg2+ monovalent cations stimulate the enzyme activity. The molecular weight of pyruvate carboxylase as determined by gel filtration of Sepharose CL-6B is 300,000. The enzyme molecule contains biotin. The apparent Km values for pyruvate, ATP and HCO3- are 1.77, 0.19 and 0.23 mM, respectively. CoASAc, alpha-ketoglutarate and glutamate have no effect on the enzyme activity. The enzyme is inhibited by aspartate, malate, oxaloacetate and is activated by citrate, isocitrate and phosphosugars. The role of pyruvate carboxylase in methylotrophic metabolism of Ps. oleovorans is discussed.
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PMID:[Properties of pyruvate carboxylase of the facultative methylotrope Pseudomonas oleovorans]. 717 45

The total adenine nucleotide content of the mitochondrial matrix increases 3--4-fold within a few hours of birth in rat liver. This provides a mechanism for the acute postnatal regulation of pyruvate carboxylase, which is located in the matrix compartment and which requires ATP as a substrate. The sudden increase in pyruvate carboxylase activity is proposed to account for a rapid 4--5-fold increase in the gluconeogenic rate of the newborn rat.
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PMID:Acute postnatal regulation of pyruvate carboxylase activity by compartmentation of mitochondrial adenine nucleotides. 726 Jan 4

1. The effect of oleate on the subcellular distribution of ATP, 2-oxoglutarate, glutamate, citrate, malate and phosphoenolpyruvate was studied in hepatocytes from rats starved for 48 h by applying a modified digitonin method. The results markedly differ from those observed after glucagon [Siess, E. A., Brocks, D. G., Lattke, H. K., and Wieland, O. H. (1977) Biochem J. 166, 225-235]. Total cellular amounts and the distribution of ATP and 2-oxoglutarate remained unchanged. In the mitochondrial matrix glutamate was increased, while mitochondrial phospho-enolpyruvate was decreased. Citrate and malate were increased both in the mitochondrial and cytosolic space. 2. In contrast to the effect of glucagon, gluconeogenesis from dihydroxyacetone, fructose or glutamine was not stimulated by oleate. Gluconeogenesis from propionate was even inhibited by the fatty acid. 3. The stimulation by glucagon of glucose production from dihydroxyacetone or fructose was undiminished in biotin-deficient hepatocytes. Glucose formation from lactate, however, was stimulated only in biotin-substituted hepatocytes. 4. The results indicate that oleate stimulates gluconeogenesis by increasing pyruvate carboxylase activity (EC 6.4.1.1), whereas glucagon displays a more complex mode of action.
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PMID:Distinctive roles of oleate and glucagen in gluconeogenesis. 746 Sep 51

The kinetics of nucleotide binding to pyruvate carboxylase have been studied by measuring the fluorescence changes that occur on the binding and release of FTP and FDP, which are fluorescent formycin analogues of ATP and ADP. The rate constants and equilibrium binding constants for both MgFTP and MgFDP binding to pyruvate carboxylase have been determined. From the kinetics of displacement of MgFTP by MgATP and binding of MgFTP in the presence of MgATP, the rate constants of MgATP binding were estimated. A slow component to the fluorescence changes was seen to occur after the initial rapid, bimolecular binding step, when formycin nucleotides were mixed with the enzyme. HCO3- and pyruvate were shown to quench the fluorescence of enzyme-bound MgFTP, but did not affect the affinity of the enzyme for the nucleotide. Acetyl CoA reduced the affinity of the enzyme for both MgFDP and MgFTP by about 3-fold by decreasing the association rate constants (by 25%) and increasing the dissociation rate constants (by 2-fold). In the absence of Mg2+ a very rapid component to FTP binding was observed that was complete within about 3 ms, but no fast component was observed comparable to that seen in the presence of 4.5 mM MgCl2. Increasing the [Mg2+] gradually abolished this very fast component of the binding, while the amplitude of the fast component increased, although the rate constant for this component did not appear to be strongly dependent on [Mg2+]. The rate constants of the slow component of Mg.formycin nucleotide binding did not appear to be dependent on nucleotide concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Kinetics of nucleotide binding to pyruvate carboxylase. 754 19

In the adipose tissue, besides fatty acid synthesis (FA-S) from glucose, which includes several mitochondrial steps, FA-S from glutamate has been demonstrated. FA-S from glutamate takes place in the cytosol through the backward pathway of Krebs cycle (BPKC) and is due to the sequential action of (1) alanine aminotransferase (ALT, EC 2.6.1.2), which is presence of pyruvate converts glutamate to oxoglutarate; (2) isocitrate dehydrogenase (NADP) (ICDH, EC 1.1.1.42), which converts oxoglutarate to isocitrate; (3) aconitate hydratase (ACO, EC 4.2.1.3), which transforms isocitrate to citrate: and (4) ATP citrate-lyase (ATP-CL, EC 4.1.3.8), which splits citrate to yield the acetyl-CoA needed for FA-S. We studied the enzymes involved in BPKC in homogenates of human adipose tissue. In normal subjects, the cytosolic activity (mumol/min/g protein) was: ALT = 10.3 +/- 1.1, ICDH = 29.5 +/- 2.8, ACO = 2.05 +/- 0.23, and ATP-CL = 1.2 +/- 0.2. Mitochondria contained less or no activity, values being 20, 9, 11, and 0% of total for ATL, ICDH, ACO, and ATP-CL, respectively. BPKC enzymes are more active than the enzymes limiting FA-S from glucose, i.e., phosphofructokinase (EC 2.7.1.11), pyruvate carboxylase (EC 6.4.1.1), and pyruvate dehydrogenase (EC 1.2.4.1). In the obese patients, cytosolic ALT and ATP-CL were increased (12.9 +/- 0.7, P < 0.05, and 2.28 +/- 0.27, P < 0.01, respectively) compared to normal, while ICDH was not changed (ACO could not be studied). Similar changes were obtained by expressing enzyme activity per fat cell number.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Fatty acid synthesis from glutamate in the adipose tissue of normal subjects and obese patients: an enzyme study. 755 12

Pyruvate carboxylase (PC) was purified to homogeneity from an overexpressing strain of the purple photosynthetic bacterium Rhodobacter capsulatus using a rapid dye-ligand affinity chromatography procedure, in which dye-bound enzyme was specifically eluted with a low concentration of acetyl-CoA, an allosteric activator of the enzyme. The enzyme purified by this method was obtained in 75% yield with a specific activity of 40 U (mg protein)-1. In contrast, affinity chromatography on a monomeric avidin column, commonly used in the purification of biotin-containing carboxylases, resulted in a yield of < 40%, with a specific activity of 10 U (mg protein)-1. The enzyme purified by the dye-linked procedure had a subunit molecular mass of 140,000 Da and was absolutely dependent on acetyl-CoA for activity. Acetyl-CoA was also effective in protecting the enzyme from thermal denaturation. The enzyme was inhibited by 2-oxoglutarate and, to a lesser extent, L-aspartate, with sigmoidal kinetics with respect to acetyl-CoA concentration. The amino acid composition, pH optimum and kinetic constants for pyruvate, ATP and bicarbonate were determined. An N-terminal sequence of 26 residues was obtained, which was homologous to the N-terminal regions of several eukaryotic PCs, propionyl-CoA carboxylases and acetyl-CoA carboxylase.
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PMID:Acetyl-CoA-dependent pyruvate carboxylase from the photosynthetic bacterium Rhodobacter capsulatus: rapid and efficient purification using dye-ligand affinity chromatography. 758 22

The enzyme activities responsible for carboxylation reactions in cell extracts of the gastric pathogen Helicobacter pylori have been studied by H14CO3- fixation and spectrophotometric assays. Acetyl coenzyme A carboxylase (EC 6.4.1.2) and malic enzyme (EC 1.1.1.40) activities were detected, whereas pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxylase (EC 4.1.3.1) and phosphoenolpyruvate carboxykinase (EC 4.1.1.49) activities were absent. However, a pyruvate-dependent, ATP-independent, and avidin-insensitive H14CO3- fixation activity, which was shown to be due to the isotope exchange reaction of pyruvate:flavodoxin oxidoreductase (EC 1.2.7.1), was present. The purified enzyme is composed of four subunits of 47, 36, 24, and 14 kDa. N-terminal sequence analysis showed that this enzyme is related to a recently recognized group of four-subunit pyruvate:ferredoxin oxidoreductases previously known only from hyperthermophiles. This enzyme from H. pylori was found to mediate the reduction of a number of artificial electron acceptors in addition to a flavodoxin isolated from H. pylori extracts, which is likely to be the in vivo electron acceptor. Indirect evidence that the enzyme is capable of in vitro reduction of the anti-H. pylori drug metronidazole was also obtained.
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PMID:Identification of carboxylation enzymes and characterization of a novel four-subunit pyruvate:flavodoxin oxidoreductase from Helicobacter pylori. 760 66


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