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Biomedical subjects

B Sumegi

Publications and source records attributed to B Sumegi.

At least 19 recordsLinked to original sources
PubMed2005

Cloning, sequencing, structural and molecular biological characterization of placental protein 20 (PP20)/human thiamin pyrophosphokinase (hTPK).

Full-length cDNAs of placental protein 20 (PP20) were cloned by screening a human placental cDNA library, which encode a 243 amino acid protein, identical to human thiamin pyrophosphokinase (hTPK) as confirmed by protein sequence analysis. Genomic alignment showed that the PP20/hTPK gene contains 9 exons. It is abundantly expressed in placenta, as numerous EST clones were identified. As thiamine metabolism deficiencies have been seen in placental infarcts previously, these indicate that PP20/hTPK may have a role in placental diseases. Analysis of the 1kb promoter region showed numerous putative transcription factor binding sites, which might be responsible for the ubiquitous PP20/hTPK expression. This may also be in accordance with the presence of the protein in tissues responsible for the regulation of the exquisite balance between cell division, differentiation and survival. TPK activity of the purified and recombinant protein was proved by mass spectrometry with electrospray ionization. By Western blot, PP20/hTPK was found in all human normal and tumorous adult and fetal tissues in nearly equal amounts, but not in sera. By immunohistochemical and immunofluorescent confocal imaging methods, diffuse labelling in the cytoplasm of the syncytiotrophoblasts and weak staining of the trophoblasts were observed, and the amount of PP20/hTPK decreased from the first trimester to the end of gestation. A 3D model of PP20/hTPK was computed (PDB No.: 1OLY) by homology modelling. A high degree of structural homology showed that the thiamin binding site was highly similar to that of the mouse enzyme, but highly different from the bacterial ones. Comparison of the catalytic centre sequences revealed differences, raising the possibility of designing new drugs which specifically inhibit bacterial and fungal enzymes without affecting PP20/hTPK and offering the possibility for safe antimicrobial therapy during pregnancy.

Adult↗
PubMed2005

Extremely high maternal alkaline phosphatase serum concentration with syncytiotrophoblastic origin.

An extremely high alkaline phosphatase (AP) concentration (3609 IU/litre) was found in a 20 year old primigravida at 37 week's gestation, prompting an examination of its histological and cellular origin. Immunohistochemistry and western blots using antibodies against AP, Ki-67, phospho-protein kinase B (Akt), phospho-p44/42 mitogen activated protein kinase/extracellular signal regulated kinase 1/2 (MAPK/Erk1/2), phospho-glycogen synthase kinase-3beta (GSK-3beta), phospho-stress activated protein kinase/c-Jun N-terminal kinase, total-Akt, total-GSK-3beta, and phospho-p38-MAPK were carried out on index and control placental samples of the same gestational age. Compared with controls, staining of the index placenta showed minimal AP labelling of the brush border and remarkable positivity of the intervillous space. Cytotrophoblastic proliferation was 8-10% in the index placenta compared with 1-2% in controls. The index placenta also had raised concentrations of protein kinases with important roles in cell differentiation. The proliferation and differentiation rates of the cytotrophoblasts were found to be five times higher in index samples than in controls. It is hypothesised that loss of syncytial membranes in immature villi led to increased AP concentrations in the maternal circulation and decreased AP staining of the placenta. Loss of the syncytium might also stimulate increased proliferation of villous cytotrophoblasts, which would then fuse and maintain the syncytium.

Adult↗
PubMed2001

Direct effect of Taxol on free radical formation and mitochondrial permeability transition.

To elucidate the potential role of mitochondria in Taxol-induced cytotoxicity, we studied its direct mitochondrial effects. In Percoll-gradient purified liver mitochondria, Taxol induced large amplitude swelling in a concentration-dependent manner in the microM range. Opening of the permeability pore was also confirmed by the access of mitochondrial matrix enzymes for membrane impermeable substrates in Taxol-treated mitochondria. Taxol induced the dissipation of mitochondrial membrane potential (DeltaPsi) determined by Rhodamine123 release and induced the release of cytochrome c from the intermembrane space. All these effects were inhibited by 2.5 microM cyclosporine A. Taxol significantly increased the formation of reactive oxygen species (ROS) in both the aqueous and the lipid phase as determined by dihydrorhodamine123 and resorufin derivative. Cytochrome oxidase inhibitor CN(-), azide, and NO abrogated the Taxol-induced mitochondrial ROS formation while inhibitors of the other respiratory complexes and cyclosporine A had no effect. We confirmed that the Taxol-induced collapse of DeltaPsi and the induction of ROS production occurs in BRL-3A cells. In conclusion, Taxol-induced adenine nucleotide translocase-cyclophilin complex mediated permeability transition, and cytochrome oxidase mediated ROS production. Because both cytochrome c release and mitochondrial ROS production can induce suicide pathways, the direct mitochondrial effects of Taxol may contribute to its cytotoxicity.

Animals↗
PubMed2001

The effect of carvedilol on enhanced ADP-ribosylation and red blood cell membrane damage caused by free radicals.

OBJECTIVE: Previous studies have reported that the beta and alpha adrenoceptor blocker carvedilol has unique protective effects on free radical-induced myocardial injury. The aim of this study was to examine how carvedilol regulates reactive-oxygen-species-mediated signaling and decreases red blood cell membrane damage in heart perfusion and in a rheological model. METHODS: The ischemia-reperfusion-induced oxidative cell damage, and changes in the intracellular signaling mediated by reactive oxygen species and peroxynitrite were studied on rat hearts in a Langendorff perfusion system (n=15). The effect of carvedilol on red blood cell suspension viscosity (hematocrit: 60%) incubated with free radical generator (phenazine methosulphate) was also investigated (n=10). The measurements were performed on a capillary viscosimeter. RESULTS: In both studies a protective effect of carvedilol was found, as the decrease of red blood cell suspension viscosity and K(+) concentration in the supernatant indicated. Carvedilol significantly decreased the ischemia-reperfusion-induced free radical production and the NAD(+) catabolism and reversed the poly- and mono(ADP-ribosyl)ation. Carvedilol also decreased the lipid peroxidation and membrane damages as determined by free malondialdehyde production and the release of intracellular enzymes. The self ADP-ribosylation of isolated poly(ADP-ribose) polymerase was also significantly inhibited by carvedilol. CONCLUSION: Our results show that carvedilol can modulate the reactive-oxygen-species-induced signaling through poly- and mono(ADP-ribosyl)ation reactions, the NAD(+) catabolism in postischemic perfused hearts and has a marked scavenger effect on free radical generator-induced red blood cell membrane damage. All these findings may play an important role in the beneficial effects of carvedilol treatment in different cardiovascular diseases.

ADP Ribose Transferases↗
PubMed2001

Effect of poly(ADP-ribose) polymerase inhibitors on the ischemia-reperfusion-induced oxidative cell damage and mitochondrial metabolism in Langendorff heart perfusion system.

Ischemia-reperfusion induces reactive oxygen species (ROS) formation, and ROS lead to cardiac dysfunction, in part, via the activation of the nuclear poly(ADP-ribose) polymerase (PARP, called also PARS and ADP-RT). ROS and peroxynitrite induce single-strand DNA break formation and PARP activation, resulting in NAD(+) and ATP depletion, which can lead to cell death. Although protection of cardiac muscle by PARP inhibitors can be explained by their attenuating effect on NAD(+) and ATP depletion, there are data indicating that PARP inhibitors also protect mitochondria from oxidant-induced injury. Studying cardiac energy metabolism in Langendorff heart perfusion system by (31)P NMR, we found that PARP inhibitors (3-aminobenzamide, nicotinamide, BGP-15, and 4-hydroxyquinazoline) improved the recovery of high-energy phosphates (ATP, creatine phosphate) and accelerated the reutilization of inorganic phosphate formed during the ischemic period, showing that PARP inhibitors facilitate the faster and more complete recovery of the energy production. Furthermore, PARP inhibitors significantly decrease the ischemia-reperfusion-induced increase of lipid peroxidation, protein oxidation, single-strand DNA breaks, and the inactivation of respiratory complexes, which indicate a decreased mitochondrial ROS production in the reperfusion period. Surprisingly, PARP inhibitors, but not the chemically similar 3-aminobenzoic acid, prevented the H(2)O(2)-induced inactivation of cytochrome oxidase in isolated heart mitochondria, suggesting the presence of an additional mitochondrial target for PARP inhibitors. Therefore, PARP inhibitors, in addition to their important primary effect of decreasing the activity of nuclear PARP and decreasing NAD(+) and ATP consumption, reduce ischemia-reperfusion-induced endogenous ROS production and protect the respiratory complexes from ROS induced inactivation, providing an additional mechanism by which they can protect heart from oxidative damages.

Aminobenzoates↗
PubMed2001

[The effect of carvedilol on enhanced adp-ribosylation and red blood cell membrane damage caused by free radicals].

OBJECTIVE: Previous studies have reported that the beta and alpha adrenoceptor blocker carvedilol had unique protective effect on free radical induced myocardial injury. The aim of this study was to examine how carvedilol regulates ROI-mediated signaling and decreases RBC membrane damage in heart perfusion and rheological model. METHODS: The ischemia-reperfusion induced oxidative cell damages, and changes in the intracellular signaling mediated by reactive oxygen species and peroxynitrite were studied on rats, in Langendorff heart perfusion system (n = 15). The effect of carvedilol on red blood cell suspension viscosity (hematocrit: 60%) incubated with free radical generator (phenazine methosulphate) was also investigated (n = 10). The measurements were performed on a capillary viscosimeter. RESULTS: In both studies a protective effect of carvedilol was found, as the decrease of red blood cell suspension viscosity and K+ concentration in the supernatant indicated. Carvedilol significantly decreased the ischemia-reperfusion induced free radical production and the NAD+ catabolism and reversed the poly- and mono-ADP-ribosylation. Carvedilol also decreased the lipid peroxidation and membrane damages as determined by free malondialdehyde production and the release of intracellular enzymes. The self ADP-ribosylation of isolated PARP was also significantly inhibited by carvedilol. CONCLUSION: Our results show that carvedilol can modulate the ROI-induced signaling through poly- and mono-ADP-ribosylation reactions, the NAD+ catabolism in postischemic perfused hearts and has a marked scavenger effect on free radical generator induced red blood cell membrane damage. All these findings may play an important role in the beneficial effects of carvedilol treatment in different cardiovascular diseases.

Adenosine Diphosphate Ribose↗
PubMed2000

BGP-15, a nicotinic amidoxime derivate protecting heart from ischemia reperfusion injury through modulation of poly(ADP-ribose) polymerase.

The protective effect of O-(3-piperidino-2-hydroxy-1-propyl)nicotinic amidoxime (BGP-15) against ischemia-reperfusion-induced injury was studied in the Langendorff heart perfusion system. To understand the molecular mechanism of the cardioprotection, the effect of BGP-15 on ischemic-reperfusion-induced reactive oxygen species (ROS) formation, lipid peroxidation single-strand DNA break formation, NAD(+) catabolism, and endogenous ADP-ribosylation reactions were investigated. These studies showed that BGP-15 significantly decreased leakage of lactate dehydrogenase, creatine kinase, and aspartate aminotransferase in reperfused hearts, and reduced the rate of NAD(+) catabolism. In addition, BGP-15 dramatically decreased the ischemia-reperfusion-induced self-ADP-ribosylation of nuclear poly(ADP-ribose) polymerase(PARP) and the mono-ADP-ribosylation of an endoplasmic reticulum chaperone GRP78. These data raise the possibility that BGP-15 may have a direct inhibitory effect on PARP. This hypothesis was tested on isolated enzyme, and kinetic analysis showed a mixed-type (noncompetitive) inhibition with a K(i) = 57 +/- 6 microM. Furthermore, BGP-15 decreased levels of ROS, lipid peroxidation, and single-strand DNA breaks in reperfused hearts. These data suggest that PARP may be an important molecular target of BGP-15 and that BGP-15 decreases ROS levels and cell injury during ischemia-reperfusion in the heart by inhibiting PARP activity.

ADP Ribose Transferases↗
PubMed1999

Molecular mechanism of the short-term cardiotoxicity caused by 2',3'-dideoxycytidine (ddC): modulation of reactive oxygen species levels and ADP-ribosylation reactions.

The short-term cardiac side effects of 2',3'-dideoxycytidine (ddC, zalcitabine) were studied in rats in order to understand the biochemical events contributing to the development of ddC-induced cardiomyopathy. In developing animals, ddC treatment provoked a surprisingly rapid appearance of cardiac malfunctions characterized by prolonged RR, PR, and QT intervals and J point depression. The energy metabolism in the heart was compromised, characterized by a decreased creatine phosphate/creatine ratio (from 2.05 normal value to 0.75) and a decreased free ATP/ADP ratio (from 332 normal value to 121). The activity of respiratory complexes (NADH: cytochrome c oxidoreductase and cytochrome oxidase) also decreased significantly. Southern blot and polymerase chain reaction analysis did not show deletions or a decrease in the quantity of mitochondrial DNA (mtDNA) deriving from ddC-treated rat hearts, indicating that under our experimental conditions, ddC-induced heart abnormalities were not the direct consequence of mtDNA-related damage. The ddC treatment of rats significantly increased the formation of reactive oxygen species (ROS) in heart and skeletal muscle as determined by the oxidation of non-fluorescent dihydrorhodamine123 to fluorescent rhodamine123 and the oxidation of cellular proteins determined from protein carbonyl content. An activation of the nuclear poly-(ADP-ribose) polymerase (EC 2.4.2.30) and an increase in the mono-ADP-ribosylation of glucose-regulated protein and desmin were observed in the cardiac tissue from ddC-treated animals. A decrease in the quantity of heat shock protein (HSP)70s was also detected, while the level of HSP25 and HSP60 remained unchanged. Surprisingly, ddC treatment induced a skeletal muscle-specific decrease in the quantity of three proteins, one of which was identified by N-terminal sequencing as myoglobin, and another by tandem mass spectrometer sequencing as triosephosphate isomerase (EC 5.3.1.1). These data show that the short term cardiotoxicity of ddC is partially based on ROS-mediated signalling through poly- and mono-ADP-ribosylation reactions and depression of HSP70 levels, whose processes represent a new mtDNA independent mechanism for ddC-induced cell damage.

Adenosine Diphosphate Ribose↗
PubMed1999

Role of reactive oxygen species and poly-ADP-ribose polymerase in the development of AZT-induced cardiomyopathy in rat.

The short term cardiac side-effects of AZT (3'-azido-3'-deoxythymidine, zidovudine) was studied in rats to understand the biochemical events contributing to the development of AZT-induced cardiomyopathy. Developing rats were treated with AZT (50 mg/kg/day) for 2 wk and the structural and functional changes were monitored in the cardiac muscle. AZT treatment provoked a surprisingly fast appearance of cardiac malfunctions in developing animals characterized by prolonged RR, PR and QT intervals and J point depression. Electron microscopy showed abnormal mitochondrial structure but the cardiomyocyte had normal myofibers. The AZT treatment of rats significantly increased ROS and peroxynitrite formation in heart tissues as determined by the oxidation of nonfluorescent dihydrorhodamine123 and dichlorodihydro-fluorescein diacetate (H2DCFDA) to fluorescent dyes, and induced single-strand DNA breaks. Lipid peroxidation and oxidation of cellular proteins determined from protein carbonyl content were increased as a consequence of AZT treatment. Activation of the nuclear poly-ADP-ribose polymerase and the accelerated NAD+ catabolism were also observed in AZT-treated animals. Western blot analysis showed that mono-ADP-ribosylation of glucose regulated protein (GRP78/BIP) was enhanced by AZT treatment, that process inactivates GRP78. In this way moderate decrease in the activity of respiratory complexes was detected in the heart of AZT-treated animals indicating a damaged mitochondrial energy production. There was a significant decrease in creatine phosphate concentration resulting in a decrease in creatine phosphate/creatine ratio from 2.08 to 0.58. ATP level remained close to normal but the total extractable ADP increased with 45%. The calculated free ATP/ADP ratio decreased from 340 to 94 in the heart of AZT-treated rats as a consequence of increased free ADP concentration. It was assumed that the increased free ADP in AZT-treated cardiomyocyte may help cells to compensate the defective ATP production in damaged mitochondria by activating the ATP synthesis in undamaged mitochondria. Southern blot analysis did not show decreased quantity of mtDNA deriving from AZT-treated rat hearts indicating that under our experimental conditions AZT-induced heart abnormalities are not the direct consequence of the mtDNA depletion. These data show that ROS-mediated oxidative damages, activated ADP-ribosylation reactions and accelerated NAD+ catabolism play basic roles in the development of AZT-induced cardiomyopathy in our animal model and indicated that these ROS-mediated processes can be important factors in the development of myopathy and cardiomyopathy in zidovudine-treated AIDS patients.

ADP Ribose Transferases↗
PubMed1999

Enhanced ADP-ribosylation and its diminution by lipoamide after ischemia-reperfusion in perfused rat heart.

Poly-ADP-ribose polymerase (PARP) is considered to play an important role in oxidative cell damage. We assumed that ischemia-reperfusion resulting from the increasing reactive oxygen species (ROS) can lead to the activation of endogenous mono- and poly-ADP-ribosylation reactions and that the reduction of ROS level by lipoamide, a less known antioxidant, can reverse these unfavorable processes. Experiments were performed on isolated Langendorff hearts subjected to 60-min ischemia followed by reperfusion. ROS, malondialdehyde, deoxyribonucleic acid (DNA) breaks, and NAD+ content were assayed in the hearts, and the ADP-ribosylation of cytoplasmic and nuclear proteins were determined by Western blot assay. Ischemia-reperfusion caused a moderate (30.2 +/- 8%) increase in ROS production determined by the dihydrorhodamine 123 method and significantly increased the malondialdehyde production (from < 1 to 23 +/- 2.7 nmol/ml), DNA damage (undamaged DNA decreased from 71 +/- 7% to 23.1 +/- 5%), and NAD+ catabolism. In addition, ischemia-reperfusion activated the mono-ADP-ribosylation of GRP78 and the self-ADP-ribosylation of the nuclear PARP. The perfusion of hearts with lipoamide significantly decreased the ischemia-reperfusion-induced cell membrane damage determined by enzyme release (LDH, CK, and GOT), decreased the ROS production, reduced the malondialdehyde production to 5.5 +/- 2.4 nmol/ml, abolished DNA damage, and reduced NAD+ catabolism. The ischemia-reperfusion-induced activation of poly- and mono-ADP-ribosylation reactions were also reverted by lipoamide. In isolated rat heart mitochondria, dihydrolipoamide was found to be a better antioxidant than dihydrolipoic acid. Ischemia-reperfusion by ROS overproduction and increasing DNA breaks activates PARP leading to accelerated NAD+ catabolism, impaired energy metabolism, and cell damage. Lipoamide by reducing ROS levels halts PARP activation and membrane damage and improves the recovery of postischemic myocardium.

ADP Ribose Transferases↗
PubMed1999

Isolation and sequence analysis of a cDNA encoding human placental tissue protein 13 (PP13), a new lysophospholipase, homologue of human eosinophil Charcot-Leyden Crystal protein.

Expression of placental tissue protein 13 (PP13) in different human tissues was investigated by chemiluminescence Western blot analysis using monospecific anti-PP13 serum. In term placentae we detected a 16 kDa single protein band immunochemically identical to the purified PP13 antigen. After investigation of 26 types of human fetal and adult tissue, PP13 was also found in certain other normal and tumorous tissue extracts. It is not secreted into circulation as we could not find PP13 in sera of pregnant women. A full length cDNA with 578 bp insert was isolated by screening a human placental cDNA library with anti-PP13 serum. The open reading frame of the cDNA encodes for a 139-residue-long protein with a predicted molecular mass of 16.118 kDa, identical to the previously isolated and characterized PP13 antigen described in 1983. By alignment search of the protein databank PP13 is highly homologous (69 per cent) to the 16.5 kDa human eosinophil Charcot-Leyden Crystal protein, a unique dual-function lysophospholipase, a member of the beta-galactoside binding S-type animal lectin superfamily. Northern blot analysis revealed a 600 bp PP13 mRNA, detected only in placental tissue from 16 types of human healthy adult tissue. Lysophospholipase activity of PP13 was confirmed by(1)H and(31)P nuclear magnetic resonance (NMR) measurements.

Adult↗
PubMed1998

Cloning and sequence analysis of cDNAs encoding human placental tissue protein 17 (PP17) variants.

Using monospecific anti-PP17 serum with chemiluminescence Western-blot analysis, we detected different molecular-mass variants of human soluble placental tissue protein 17 (PP17) in different normal adult and fetal human tissues besides term placenta. 13 cDNAs with three different insert lengths encoding PP17 variants were isolated by screening a human placental cDNA library. Sequence analysis of the shortest clones showed that the inserts contain the same open reading frame encoding PP17a variant (28,129 kDa) consisting of 251 residues, which is identical to the previously isolated and characterised PP17 antigen described in 1983. The ubiquitous PP17b variant is encoded by longer clones and contains 434 residues with a predicted molecular mass of 47,208 kDa. Compared to normal conditions, these newly discovered PP17 variants are overexpressed in cervix carcinoma tissue, as are their three different-size messenger RNAs in HeLa cell line. Increased amounts of PP17b are secreted into the circulation in cervix carcinoma patients. We also observed a typical elevation in serum levels of PP17 variants during healthy pregnancy. An alignment search of the protein databank showed that PP17a and PP17b are homologous to adipose tissue differentiation and lipid-droplet-associated proteins: human adipophilin, mouse adipose differentiation-related protein and rat perilipin A and B.

Amino Acid Sequence↗
PubMed1998

Autoantibodies against subunits of pyruvate dehydrogenase and citrate synthase in a case of paediatric biliary cirrhosis.

In a newborn girl with a history of connatal liver damage, histological examination of a liver biopsy sample taken during the seventh week of life revealed incipient destruction of bile ducts. Very high titres of antimitochondrial antibodies were later detected in the plasma. As the hepatic injury tended towards fibrosis, the histological diagnosis became primary biliary cirrhosis. Autoantibodies against E1 alpha, E2, and E3 subunits and protein X component of pyruvate dehydrogenase complex, and against citrate synthase were detected on western immunoblotting in a 1 in 1000 dilution of the patient's serum. The patient died of her illness at 11 years of age. In liver specimens obtained at autopsy human immunoglobulin deposition was detected on the surface of almost all hepatic cells by immunohistology. As there is a physical and functional interaction between pyruvate dehydrogenase and citrate synthase within the mitochondria, the presence of autoantibodies against certain proteins in the patient suggests that in this form of the disease the molecular recognition and then the autoimmunisation process could be directed against a mitochondrial enzyme cluster containing both pyruvate dehydrogenase and citrate synthase.

Autoantibodies↗
PubMed1995

Metabolism of [3-13C]pyruvate and [3-13C]propionate in normal and ischaemic rat heart in vivo: 1H- and 13C-NMR studies.

The oxidation of [3-13C]pyruvate and [3-13C]propionate was studied in vivo in infused rats. The infused [3-13C]pyruvate was quickly converted to [3-13C]lactate in the blood, and the [3-13C]lactate formed was well metabolized in both normoxic and ischaemic hearts. Large differences (200-600%) in the 13C enrichment of alanine (C-3) and acetyl-CoA (C-2) compared with lactate (C-3) were found in both normoxic and ischaemic hearts, suggesting that the extracellular [3-13C]lactate preferentially entered a region of the cytoplasm which specifically transfers the labelled pyruvate (formed from [3-13C]lactate) to the mitochondria. The highly enriched mitochondrial pyruvate gave high enrichment in alanine and acetyl-CoA, which was detected by 1H- and 13C-NMR spectroscopy. Ischaemia increased 13C incorporation into the main cytoplasmic lactate pool and decreased 13C incorporation into citric acid cycle intermediates, mainly decreasing the pyruvate anaplerosis. Isoprenaline-induced ischaemia of the heart caused only a slight decrease in pyruvate oxidation. In contrast to the decreased anaplerosis of pyruvate, the anaplerosis of propionate (and propionyl-carnitine) increased significantly in ischaemic hearts, which may contribute to the protective effect of propionyl-carnitine seen in ischaemia. In addition, we found that [3-13C]propionate preferentially labelled aspartate C-3 in rat heart, suggesting incomplete randomization of label in the succinyl-CoA-malate span of the citric acid cycle. These data show that proton observed 13C edited spectroscopic methods, i.e. heteronuclear spin-echo and the one-dimensional heteronuclear multiple quantum coherence sequence, can be successfully used to study heart metabolism in vivo.

Acetyl Coenzyme A↗
PubMed1994

Orientation-conserved transfer of symmetric Krebs cycle intermediates in mammalian tissue.

Metabolism of [2-13C]-, [3-13C]-, and [1,2,3-13C]propionate in perfused rat livers and [2-13C]-acetate in perfused rat hearts has been examined in tissue extracts by 13C NMR. Label from [2-13C]-propionate was preferentially incorporated into the C2 carbon of lactate, alanine, and aspartate in liver tissue while label from [3-13C]propionate appeared preferentially in the C3 carbon of those same molecules. These data suggest that 13C may not be completely randomized in the symmetric citric acid cycle intermediates succinate and fumarate as is normally assumed but that some fraction of those intermediates may be transferred between enzymes in this span of the cycle with conservation of spatial orientation, consistent with recent results obtained in yeast [Sumegi et al. (1990) Biochemistry 29, 9106-9110]. This was confirmed by performing similar experiments with [1,2,3-13C]propionate. Time-dependent asymmetry was also observed between the intensities of the glutamate C2 and C3 resonances and between the aspartate C2 and C3 resonances in 13C NMR spectra of intact hearts and heart extracts during early perfusion with [2-13C]-acetate. A model is presented which predicts that isotopic asymmetry is observed only during the first 2-3 turns of the cycle pools when isotope enters the cycle via acetyl-CoA even if all symmetric cycle intermediates retain a unique molecular orientation on each pass through the citric acid cycle.

Acetates↗
PubMed1994

Lipoamide influences substrate selection in post-ischaemic perfused rat hearts.

We investigated whether lipoamide and diacetyl-lipoamide are able to change the substrate selection in post-ischaemic myocardium. This can be important, because shifting heart metabolism from fatty acid to carbohydrate oxidation can decrease ischaemic injury. Studying the metabolism of [1,2-13C]diacetyl-lipoamide in situ in perfused rat heart by 13C n.m.r., we found intense 13C labelling in glutamate and aspartate, showing that acetyl groups from diacetyl-lipoamide are effectively transferred to CoA and metabolized in heart tissue. From analysis of glutamate C-3 and C-4 isotopomers, we determined the [1,2-13C]acetate/[3-13C]lactate utilization ratio in normoxic and post-ischaemic hearts, where under our experimental conditions the acetate/lactate utilization ratios were 1.2 +/- 0.2 and 2.4 +/- 0.3 in normoxic and post-ischaemic hearts respectively. When 0.25 mM lipoamide was added to the perfusate the acetate/lactate utilization ratio decreased to 1.4 +/- 0.1, which is almost equal to that found for normoxic hearts, showing that lipoamide increased the lactate utilization. In accordance with these data, we found that lipoamide activated pyruvate dehydrogenase by 50% in post-ischaemic myocardium. Competition between [3-13C]lactate and unlabelled octanoate was also studied in post-ischaemic hearts, and we found that lipoamide increased lactate utilization by 100% and increased the rate of the tricarboxylic acid cycle by 64%. Under the same experimental conditions, lipoamide significantly promoted the recovery of post-ischaemic unpaced hearts, showing the positive effect of increased lactate oxidation in post-ischaemic myocardium.

Acetates↗
PubMed1993

Evidence for orientation-conserved transfer in the TCA cycle in Saccharomyces cerevisiae: 13C NMR studies.

When [4-13C]glutamate is incubated with yeast cells, the [13C]aspartate formed shows a [2-13C]/[3-13C] ratio that is greater than 1. If the conversion occurs through the Krebs TCA cycle with the symmetrical intermediates succinate and fumarate, the ratio is expected to be 1. The addition of fluoroacetate to the incubation increases the ratio further. When the mutants Aco1- or CS1- are used, little or no aspartate is formed. When [2-13C]acetate is incubated with yeast, the C2-/C3-aspartate ratio is 1.5 at 2 min and decrease to 1.0 after 10 min of incubation. These results indicate that orientation-conserved transfer occurs at the succinate thiokinase and succinate dehydrogenase steps of the Krebs TCA cycle.

Acetates↗