Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A causative factor in the development of diabetes-induced heart dysfunction may be abnormalities in myocardial energy metabolism. Using 13C-NMR spectroscopy, we investigated the effects of experimentally induced diabetes (streptozotocin 65 mg/kg, i.v.) on glucose metabolism and contractile function in the isolated perfused rat heart. Hearts from streptozotocin-treated and untreated control rats were perfused with 11 mM [1-13C]glucose as substrate and 1H-decoupled 13C-spectra recorded for up to 90 min. Incorporation of label from [1-13C]glucose into lactate and glutamate was observed in hearts from control animals, consistent with metabolism through glycolysis and TCA cycle, respectively. Diabetic hearts did not incorporate label into lactate or glutamate. Addition of insulin (0.05 U/ml) to the buffer resulted in the appearance of [3-13C]lactate, although glutamate labeling was not observed. Addition of insulin plus dichloroacetate (2 mM) resulted in incorporation of label from [1-13C]glucose into 2-, 3- and 4-13C-glutamate, indicating glucose entry into the TCA cycle. Addition of insulin, or insulin plus dichloroacetate to control hearts did not alter labeling of either lactate or glutamate. Cardiac function in hearts from the diabetic group was depressed compared to controls and declined significantly over the duration of the experiment. These studies show that concomitant with a decrease in cardiac function, glucose oxidation is profoundly inhibited following the induction of diabetes with streptozotocin. These observations are consistent with a combination of decreased glucose transport and a decrease in pyruvate dehydrogenase activity.
J Mol Cell Cardiol 1993 Oct
PMID:A 13C-NMR study of glucose oxidation in the intact functioning rat heart following diabetes-induced cardiomyopathy. 826 54

DEAE-cellulose chromatography of extracts of free-living Rhizobium meliloti cells revealed separate NAD(+)-dependent and NADP(+)-dependent malic enzyme activities. The NAD+ malic enzyme exhibited more activity with NAD+ as cofactor, but also showed some activity with NADP+. The NADP+ malic enzyme only showed activity when NADP+ was supplied as cofactor. Three independent transposon-induced mutants of R. meliloti which lacked NAD+ malic enzyme activity (dme-) but retained NADP+ malic enzyme activity were isolated. In an otherwise wild-type background, the dme mutations did not alter the carbon utilization phenotype; however, nodules induced by these mutants failed to fix N2. Structurally, these nodules appeared to develop like wild-type nodules up to the stage where N2-fixation would normally begin. These results support the proposal that NAD+ malic enzyme, together with pyruvate dehydrogenase, functions in the generation of acetyl-CoA required for TCA cycle function in N2-fixing bacteroids which metabolize C4-dicarboxylic acids supplied by the plant.
Mol Microbiol 1993 Mar
PMID:NAD(+)-dependent malic enzyme of Rhizobium meliloti is required for symbiotic nitrogen fixation. 838 44

Since acetyl-CoA produced through pyruvate dehydrogenase reaction is poorly oxidized by the Krebs cycle in rat lymphocytes, the fate of acetyl units was investigated in these cells. The results presented here show that 24-h cultured lymphocytes actively synthesize lipids from [3-14C]pyruvate. Furthermore, a considerable amount of these lipids have shown to be exported into the culture medium. Experiments with [1-14C] acetate as a lipid precursor showed a close similarity with the rates of incorporation of [3-14C] pyruvate into the same lipid fractions. Treatment of lymphocytes with the mitogen concanavalin A (Con A) markedly enhanced [1-14C] acetate incorporation into a variety of lipids, but the lectin did not affect [3-14C] pyruvate incorporation. The results suggest that lymphocytes convert pyruvate into lipids via the acetyl-CoA pathway and that Con A interferes in lymphocyte lipogenesis but does not seem to affect the pyruvate dehydrogenase reaction. The ability to incorporate pyruvate into certain lipids may have an important role for the rapidly dividing capacity of lymphocytes since the human cancer strain HeLa 155 (a quickly proliferating cell line) also exhibits this feature by converting much more [3-14C] pyruvate into lipids than do lymphocytes. In addition, comparative experiments with lymphocytes, peritoneal macrophages and HeLa cells indicate that pyruvate may provide precursors for cells with active lipid producing and exporting capacities.
Biochem Mol Biol Int 1993 Jul
PMID:Pyruvate is a lipid precursor for rat lymphocytes in culture: evidence for a lipid exporting capacity. 840 20

The structure of the lipoyl domain from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been determined by means of nuclear magnetic resonance spectroscopy. A total of 452 nuclear Overhauser effect distance constraints and 76 dihedral angle restraints were employed as the input for the structure calculations, which were performed using a hybrid distance geometry-simulated annealing strategy and the programs DISGEO and X-PLOR. The overall structure of the lipoyl domain (residues 1 to 79 of the dihydrolipoamide acetyltransferase polypeptide chain) is that of a flattened eight-stranded beta-barrel folded around a core of well-defined hydrophobic residues. The lipoylation site, lysine 42, is located in the middle of a beta-turn, and the N and C-terminal residues of the domain are close together in adjacent beta-strands at the opposite end of the molecule. The polypeptide backbone exhibits a 2-fold axis of quasi-symmetry, with the C alpha atoms of residues 15 to 39 and 52 to 76 being almost superimposable on those of residues 52 to 76 and 15 to 39, respectively (root-mean-square deviation = 1.48 A). The amino acid residues at key positions in the structure are conserved among all the reported primary structures of lipoyl domains, suggesting that the domains all fold in a similar way.
J Mol Biol 1993 Feb 20
PMID:Three-dimensional structure of the lipoyl domain from Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex. 844 35

The three-dimensional structure of a 43-residue active, synthetic peptide encompassing the peripheral subunit-binding domain of dihydrolipoamide acetyltransferase from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been determined by means of a multi-cooling dynamical simulated annealing protocol using restraints derived from 1H nuclear magnetic resonance spectroscopy. A total of 442 experimentally derived restraints including 13 dihedral angle (phi, chi 1) restraints were used. A final set of 35 structures was calculated with a root-mean-square deviation from the mean co-ordinates of 0.36 A for the backbone atoms and 0.96 A when side-chain heavy atoms were included for the well-defined region comprising residues Val7 to Leu39. Although assignments were made and sequential connectivities observed for the N-terminal six and C-terminal four residues, the absence of long-range NOEs suggests that the terminal regions are largely unstructured. The binding domain contains two short parallel alpha-helices (residues Val7 to Lys14 and Lys32 to Leu39), a3(10)-helix (residues Asp17 to Val21) and a structured loop made up of overlapping beta-turns (residues Gln22 to Leu31), which enclose a close-packed hydrophobic core. The loop is stabilized to a large extent by Asp34. This residue is conserved in all peripheral subunit-binding domains and its carboxylate side-chain forms a set of side-chain-main-chain hydrogen bonds with the main-chain amide protons of Gly23, Thr24, Gly25 and Leu31 and a side-chain-side-chain hydrogen bond with the hydroxyl group of Thr24. We propose that a peripheral subunit-binding site may be located in the loop region, which contains a series of highly conserved residues and provides a number of potential recognition sites. The structured region of the binding domain, comprising 33 residues, represents an exceptionally short amino acid sequence with defined tertiary structure that has no disulphide bond, ligand or cofactor to stabilize the fold. It may be approaching the lower size limit for a three-dimensional structure possessing features characteristic of larger structures, including a close-packed, non-polar interior. The organization of the side-chains in the hydrophobic core may have implications for de novo protein design.
J Mol Biol 1993 Mar 05
PMID:The high-resolution structure of the peripheral subunit-binding domain of dihydrolipoamide acetyltransferase from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. 845 May 44

The effect of valproate and its more active metabolite E-delta 2-valproate on the rate of glucose oxidation through different metabolic pathways in neonatal rat brain slices was studied. The presence of valproate or E-delta 2-valproate did not change the rate of [3,4-14C]glucose or [6-14C]glucose incorporation into CO2, suggesting that glucose oxidation through the pyruvate dehydrogenase-catalyzed reaction and through the tricarboxylic acid cycle was not affected by these drugs. However, both drugs significantly enhanced the rate of [2-14C]glucose oxidation, supporting the notion that the activity of the gamma-aminobutyric acid (GABA) shunt is specifically stimulated by valproate and, to a greater extent, by E-delta 2-valproate. The presence of methionine sulfoximine or gamma-hydroxybutyrate did not change the GABA shunt activity. Brain glutamate decarboxylase activity was significantly increased after incubation of the brain slices in the presence of valproate. Consequently, our results suggest that the mechanism of action of valproate is related to the increase in the levels of the inhibitory neurotransmitter GABA caused by the enhancement of flux through the glutamate decarboxylase-catalyzed reaction.
Mol Pharmacol 1993 Mar
PMID:Evidence of stimulation of the gamma-aminobutyric acid shunt by valproate and E-delta 2-valproate in neonatal rat brain. 845 Aug 38

Dihydrolipoyl transacetylase (E2p) is both structurally and functionally the central enzyme of the pyruvate dehydrogenase multienzyme complex. The crystal structure of the catalytic domain, i.e. residues 382 to 637, of Azotobacter vinelandii E2p (E2pCD) was solved by multiple isomorphous replacement and refined by energy minimization procedures. The final model contains 2182 protein atoms and 37 ordered water molecules. The R-factor is 18.7% for 10,344 reflections between 10.0 and 2.6 A resolution. The root-mean-square shift deviation from the ideal values is 0.017 A for bond lengths and 3.3 degrees for bond angles. The N-terminal residues 382 to 394 are disordered and not visible in the electron density map, otherwise all residues have well-defined density. The catalytic domain forms an oligomer of 24 subunits, having octahedral 432 symmetry. In the E2pCD crystals, the 24 subunits are related by the crystallographic symmetry. The cubic arrangement of subunits gives rise to a large hollow cube with edges of 120 A. The faces of the cube have pores of diameter of 30 A. The true building block of the cube is the E2p trimer, eight of which occupy the corners of the cube. Two levels of intermolecular contacts can be distinguished: (1) the extensive interactions between 3-fold related subunits leading to a tightly associated trimer; and (2) the interactions along the 2-fold axis leading to the assembly of the trimers into the cubic 24-mer. Each subunit has a topology similar to chloramphenicol acetyltransferase (CAT) and comprises a central beta-sheet surrounded by five alpha-helices. The comparison of the two proteins indicates a large rotation of the N-terminal residues 395 to 426 of E2pCD, which reshapes the substrate binding site and extends the interaction between threefold related subunits. The catalytic centre consists of a 30 A long channel extending from the "inner" side of the trimer to the "outer" side, where inner and outer refer to the position in the 24-meric cubic core of the pyruvate dehydrogenase complex and correspond with CoA and lipoamide binding sites, respectively. The active site is formed by the residues with the lowest mobility as indicated by the atomic B-factors. Five proline residues surround the active site.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1993 Apr 20
PMID:Refined crystal structure of the catalytic domain of dihydrolipoyl transacetylase (E2p) from Azotobacter vinelandii at 2.6 A resolution. 848

The structure of Pseudomonas fluorescens lipoamide dehydrogenase, a dimeric flavoenzyme with a molecular mass of 106,000 daltons, was solved by the molecular replacement method and refined to an R-factor of 19.4% at 2.8 A resolution. The root-mean-square difference from ideal values for bonds and angles is 0.019 A and 3.8 degrees, respectively. The structure is closely related to that of the same flavoprotein from Azotobacter vinelandii. The root-mean-square difference for 932 C alpha atoms is 0.64 A, with 84% sequence identity. The residues in the active site are identical, while 89% of the interface residues are the same in the two enzymes. A few structural variations provide the basis for the differences in thermostability and redox properties between the two homologous proteins. Particularly, in the A. vinelandii molecule a threonine to alanine (T452A) mutation leaves a buried carbonyl oxygen, located at the subunit interface and in proximity of the flavin ring, unpaired to any H-bond donor, probably providing an explanation for the lower stability of the A. vinelandii enzyme with respect to the P. fluorescens enzyme. Six surface loops, which previously could not be accurately positioned in the A. vinelandii structure, are well defined in P. fluorescens lipoamide dehydrogenase. On the basis of the P. fluorescens structure, the six loops could be correctly defined also in the A. vinelandii enzyme. This is an unusual case where similar refinement methodologies applied to two crystal forms of closely related proteins led to electron density maps of substantially different quality. The correct definition of these surface residues is likely to be an essential step for revealing the structural basis of the interactions between lipoamide dehydrogenase and the other members of the pyruvate dehydrogenase multienzyme complex.
J Mol Biol 1993 Apr 20
PMID:Three-dimensional structure of lipoamide dehydrogenase from Pseudomonas fluorescens at 2.8 A resolution. Analysis of redox and thermostability properties. 848 1

Thiamine or vitamin B-1, is an essential constituent of all cells since it is a cofactor for two enzyme complexes involved in the citric acid cycle, pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase. Thiamine is synthesized by plants, but it is a dietary requirement for humans and other animals. The biosynthetic pathway for thiamine in plants has not been well characterized and none of the enzymes involved have been isolated. Here we report the cloning and characterization of two cDNAs representing members of the maize thi1 gene family encoding an enzyme of the thiamine biosynthetic pathway. This assignment was made based on sequence homology to a yeast thiamine biosynthetic gene and by functional complementation of a yeast strain in which the endogenous gene was inactivated. Using immunoblot analysis, the thi1 gene product was found to be located in a plastid membrane fraction. RNA gel blot analysis of various tissues and developmental stages indicated thi1 expression was differentially regulated in a manner consistent with what is known about thiamine synthesis in plants. This is the first report of cDNAs encoding proteins involved in thiamine biosynthesis for any plant species.
Plant Mol Biol 1995 Nov
PMID:Evidence for the thiamine biosynthetic pathway in higher-plant plastids and its developmental regulation. 854 6

In mammalian cells, increases in calcium concentration cause increases in oxidative phosphorylation. This effect is mediated by the activation of four mitochondrial dehydrogenases by calcium ions; FAD-glycerol 3-phosphate dehydrogenase, pyruvate dehydrogenase, NAD-isocitrate dehydrogenase and oxoglutarate dehydrogenase. FAD-glycerol 3-phosphate dehydrogenase, being located on the outer surface of the inner mitochondrial membrane, is exposed to fluctuations in cytoplasmic calcium concentration. The other three enzymes are located within the mitochondrial matrix. While the kinetic properties of all of these enzymes are well characterised, the molecular basis for their regulation by calcium is not. This review uses information derived from calcium binding studies, analysis of conserved calcium binding motifs and comparison of amino acid sequences from calcium sensitive and non-sensitive enzymes to discuss how the recent cloning of several subunits from the four dehydrogenases enhances our understanding of the ways in which these enzymes bind calcium. FAD-glycerol 3-phosphate dehydrogenase binds calcium ions through a domain which is part of the polypeptide chain of the enzyme. In contrast, it is possible that the calcium sensitivity of the other three dehydrogenases may involve separate calcium binding subunits.
Mol Cell Biochem
PMID:Towards the molecular basis for the regulation of mitochondrial dehydrogenases by calcium ions. 856 30


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