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Query: UNIPROT:P06889 (Mol)
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A study was undertaken to assess the role of a physiological concentration of glutamine in AS-30D cell metabolism. Flux of 14C-glutamine to 14CO2 and of 14C-acetate to glutamate was detected indicating reversible flux between glutamate and TCA cycle alpha-ketoglutarate. These fluxes were transaminase dependent. A flux analysis was compared using data from three tracers that label alpha-ketoglutarate carbon 5, [2-14C]glucose, [1-14C]acetate and [5-14C]glutamine. The analysis indicated that the probability of flux of TCA cycle alpha-ketoglutarate to glutamate was, at minimum, only slightly less than the probability of flux of alpha-ketoglutarate through alpha-ketoglutarate dehydrogenase. The apparent Km for oxidative flux of [14C]glutamine to 14CO2, 0.07 mM, indicated that this flux was at a maximal rate at physiological, 0.75 mM, glutamine. Although oxidative flux through alpha-ketoglutrate dehydrogenase was the major fate of glutamine, flux of glutamine to lipid via reductive carboxylation of alpha-ketoglutarate was demonstrated by measuring incorporation of [5-14C]glutamine into 14C-lipid. In media containing glucose (6 mM), and glutamine (0.75 mM) 47 per cent of the lipid synthesized from substrates in the media was derived from glutamine via reductive carboxylation and 49 per cent from glucose. These findings of nearly equal fluxes suggest that lipogenesis via reductive carboxylation may be an important role of glutamine in hepatoma cells.
Mol Cell Biochem 1995 Nov 22
PMID:Glutamine metabolism in AS-30D hepatoma cells. Evidence for its conversion into lipids via reductive carboxylation. 875 Nov 55

The major 2-oxoacid oxidoreductase (2-OR), pyruvate:ferredoxin oxidoreductase (PFOR) from Giardia duodenalis has been purified to apparent homogeneity. A second 2-OR with a preference for alpha-ketobutyrate as substrate was identified and was removed from PFOR containing fractions during purification. Only PFOR and the second 2-OR were identified in gels of crude Giardia extracts assayed for 2-OR activity. The native form of PFOR which is membrane associated, is a homodimer of 138 kDa subunits. Pyruvate is the preferred substrate: alpha-ketobutyrate and oxaloacetate, but not phenyl-pyruvate or alpha-ketoglutarate, are decarboxylated. PFOR from Giardia is more stable than PFOR from most other organisms and purified PFOR can be stored without deterioration at -70 degrees C. Purified PFOR donates electrons to Giardia ferredoxin (Fd I) with concomitant reduction of metronidazole. However, two other Giardia ferredoxins did not accept electrons from PFOR. Consistent with the involvement of PFOR in metronidazole activation, the activity of pyruvate dependent 2-OR activity was decreased in all metronidazole-resistant lines tested but not in furazolidone-resistant lines. The presence of three different ferredoxins and two 2-ORs in Giardia suggests that a number of different electron transport pathways operate in this organism providing unusual metabolic flexibility for a eukaryote.
Mol Biochem Parasitol 1996 Aug
PMID:Characterisation and purification of pyruvate:ferredoxin oxidoreductase from Giardia duodenalis. 885 55

The effects of two metabolic inhibitors, methyl palmoxirate (MP) and amino-oxyacetate (AOA), on mouse preimplantation embryo development and cell number, and inner cell mass. (ICM) cell metabolism have been examined. Two-cell embryos were cultured in media supplemented with either MP, which inhibits fatty acid oxidation, or AOA, which inhibits the transamination of glutamate into alpha-ketoglutarate. Embryos were scored for development daily. On day 5, expanded blastocysts were differentially labeled with fluorochromes to visualize TE and ICM cell nuclei, or the ICMs isolated by immunosurgery and their energy metabolism determined using microfluorometric methods. Embryos exposed to the two inhibitors developed into fully expanded blastocysts, although cell numbers of both the TE and ICM cells were significantly reduced compared to controls. The uptake of glucose in the presence of 1 mM MP or AOA did not differ from the controls, but less glucose was accountable for by lactate production. MP significantly reduced lactate production. In the presence of 4 mM AOA, the amount of glucose oxidized and the amount of lactate formed by ICMs were significantly reduced. The results indicate that the fuels used by isolated mouse ICMs vary in response to substrate availability and that fatty acids may be a potential energy source.
Mol Reprod Dev 1996 Mar
PMID:Effects of metabolic inhibitors on mouse preimplantation embryo development and the energy metabolism of isolated inner cell masses. 886 45

Glutamine is utilized as an energy substrate in preimplantation mouse embryos. Glutaminase is the enzyme responsible for the conversion of glutamine to glutamic acid, which then enters the trichloro acetic acid (TCA) cycle as alpha-ketoglutarate. Glutaminase enzyme activity was assessed in preimplantation embryos that developed in vivo, and glutaminase RNA expression was examined in embryos that developed in vivo or were cultured in CZB medium to various preimplantation stages between 1-cell and blastocyst. Glutaminase activity in 1-8-cell-stage mouse embryos that developed in vivo ranged from 0.009-0.01 U/mg protein (2.39-2.95 x 10(-7) U per embryo) and increased 3-4 fold to 0.034 U/mg protein (8.13 x 10(-7) U per embryo) at the blastocyst stage. Relative stage-specific expression of glutaminase RNA was assessed by reverse transcription polymerase chain reaction (RT-PCR) in embryos that developed both in vivo and in CZB culture. In vivo, glutaminase RNA was expressed at the 1-cell stage, declined to 23% of 1-cell levels at the early 2-cell stage, and reaccumulated from late 2-cell through blastocyst stage, where it reached a high of 204% of 1-cell levels. CZB-cultured embryos exhibited a similar pattern of developmental RNA expression, declining to 30% of 1-cell levels at the early 2-cell stage, and increasing RNA expression at the blastocyst stage to 191% of the 1-cell level.
Mol Reprod Dev 1997 Jul
PMID:Analysis of glutaminase activity and RNA expression in preimplantation mouse embryos. 917 Jan 4

The effect of methylglyoxal on the oxygen consumption of mitochondria of heart and of several other organs of normal animals of different species has been tested. The results indicate that methylglyoxal (3.5 mM) strongly inhibits ADP-stimulated alpha-oxoglutarate and malate plus pyruvate-dependent respiration of exclusively heart mitochondria of normal animals of different species. Whereas, with the same substrates, but at a higher concentration of methylglyoxal (7.5 mM), the respiration of mitochondria of other organs of normal animals is not inhibited. Methylglyoxal also inhibits the respiration of slices of rat and toad hearts. But this inhibition is less pronounced. However, methylglyoxal (15 mM) fails to have any effect on perfused toad heart. Using rat heart mitochondria as a model, the effect of methylglyoxal on the oxygen consumption was also tested with different respiratory substrates, electron donors at different segments of the mitochondrial respiratory chain and site-specific inhibitors to identify the specific respiratory complex which might be involved in the inhibitory effect of methylglyoxal. The results strongly suggest that methylglyoxal inhibits the electron flow through complex I of rat heart mitochondrial respiratory chain. Moreover, lactaldehyde (0.6 mM), a catabolite of methylglyoxal, can exert a protective effect on the inhibition of rat heart mitochondrial respiration by methylglyoxal (2.5 mM). The effect of methylglyoxal on heart mitochondria as described in the present paper is strikingly similar to the results of our previous work with mitochondria of Ehrlich ascites carcinoma cells and leukemic leukocytes. We have recently proposed a new hypothesis on cancer which suggests that excessive ATP formation in cells may lead to malignancy. The above mentioned similarity apparently provides a solid experimental foundation for the proposed hypothesis which has been discussed.
Mol Cell Biochem 1997 Jun
PMID:Similar nature of inhibition of mitochondrial respiration of heart tissue and malignant cells by methylglyoxal. A vital clue to understand the biochemical basis of malignancy. 920 1

A 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.11) which catalyzes the 4-hydroxylation of desacetoxyvindoline was purified to homogeneity. Three oligopeptides isolated from a tryptic digest of the purified protein were microsequenced and one oligopeptide showed significant homology to hyoscyamine 6 beta-hydroxylase from Hyoscyamus niger. A 36-mer degenerate oligonucleotide based on this peptide sequence was used to screen a Catharanthus roseus cDNA library and three clones, cD4H-1 to -3, were isolated. Although none of the three clones were full-length, the open reading frame on each clone encoded a putative protein containing the sequence of all three peptides. Primer extension analysis suggested that cD4H-3, the longest cDNA clone, was missing 156 bp at the 5' end of the clone and sequencing of the genomic clone, gD4H-8, confirmed these results. Southern blot analysis suggested that d4h is present as a single-copy gene in C. roseus which is a diploid plant, and the significant differences in the sequence of the 3'-UTR between cD4H-1 and -3 suggest that they represent dimorphic alleles of the same hydroxylase. The identity of the clone was further confirmed when extracts of transformed Escherichia coli expressed D4H enzyme activity. The D4H clone encoded a putative protein of 401 amino acids with a calculated molecular mass of 45.5 kDa and the amino acid sequence showed a high degree of similarity with those of a growing family of 2-oxoglutarate-dependent dioxygenases of plant and fungal origin. The similarity was not restricted to the dioxygenase protein sequences but was also extended to the gene structure and organization since the 205 and 1720 bp introns of d4h were inserted around the same highly conserved amino acid consensus sequences as those for e8 protein, hyoscyamine-6 beta-hydroxylase and ethylene-forming enzyme. These results provide further support that a common ancestral gene is responsible for the appearance of this family of dioxygenases. Hydroxylase assays and RNA blot hybridization studies showed that enzyme activity followed closely the levels of d4h transcripts, occurring predominantly in young leaves and in much lower levels in stems and fruits. In contrast, etiolated seedlings which contained considerable levels of d4h transcripts had almost undetectable hydroxylase activity, whereas exposure of seedlings to light resulted in a rapid increase of enzyme activity without a significant further increase in d4h transcripts over those detected in dark-grown seedlings. These results suggest that the activating effect of light may occur at a point downstream of transcription which remains to be elucidated.
Plant Mol Biol 1997 Aug
PMID:Molecular cloning and characterization of desacetoxyvindoline-4-hydroxylase, a 2-oxoglutarate dependent-dioxygenase involved in the biosynthesis of vindoline in Catharanthus roseus (L.) G. Don. 929 Jun 45

We show that tetraphenylphosphonium inhibits oxidation of palmitoylcarnitine, pyruvate, malate, 2-oxoglutarate and glutamate in heart mitochondria in the range of concentration (1-5 microM) commonly used for the determination of mitochondrial membrane potential. The inhibition of 2-oxoglutarate (but not other substrate) oxidation by tetraphenylphosphonium is dependent on the concentration of 2-oxoglutarate and on extramitochondrial free calcium, and the kinetic plots are consistent with a mixed type of inhibition. Our results indicate that tetraphenylphosphonium interacts with enzymes, specifically involved in the oxidation of 2-oxoglutarate, most possibly, 2-oxoglutarate dehydrogenase.
Mol Cell Biochem 1997 Sep
PMID:Tetraphenylphosphonium inhibits oxidation of physiological substrates in heart mitochondria. 930 67

The phosphorylation state of the putative signal transduction protein P(II) from the cyanobacterium Synechococcus sp. strain PCC 7942 depends on the cellular state of nitrogen and carbon assimilation. In this study, dephosphorylation of phosphorylated P(II) protein (P[II]-P) was investigated both in vivo and in vitro. The in vivo studies implied that P(II)-P dephosphorylation is regulated by inhibitory metabolites involved in the glutamine synthetase-glutamate synthase pathway of ammonium assimilation. An in vitro assay for P(II)-P dephosphorylation was established that revealed a Mg2+-dependent P(II)-P phosphatase activity. P(II)-P phosphatase and P(II) kinase activities could be separated biochemically. A partially purified P(II)-P phosphatase preparation also catalysed the dephosphorylation of phosphoserine/phosphothreonine residues on other proteins in a Mg2+-dependent manner. However, only dephosphorylation of P(II)-P was regulated by synergistic inhibition by ATP and 2-oxoglutarate. As the same metabolites stimulate the P(II) kinase activity, it appears that the phosphorylation state of P(II) is determined by ATP and 2-oxoglutarate-dependent reciprocal reactivity of P(II) towards its phosphatase and kinase.
Mol Microbiol 1997 Oct
PMID:Dephosphorylation of the phosphoprotein P(II) in Synechococcus PCC 7942: identification of an ATP and 2-oxoglutarate-regulated phosphatase activity. 938 91

Citrate synthase which condenses acetyl-CoA and oxaloacetate to citrate was purified from Drosophila melanogaster. Some physicochemical as well as enzymatical properties were investigated. The optimum pH and temperature were pH 8.0-9.0 and 45 degrees C, respectively. The molecular weight of the enzyme was determined as 81,000 Da by gel filtration and the purified active enzyme consisted of two identical subunits which had a molecular mass of 48,700 on SDS-PAGE. Homogeneity of the purified enzyme was confirmed by SDS-PAGE and also by N-terminal amino acid sequence analysis. The Michaelis constants (K(m)) of the enzyme for acetyl-CoA and oxaloacetate were 6.7 microM and 3.1 microM, respectively. Kinetic studies showed that citrate synthase follows the concerted mechanism which forms a ternary complex. Propionyl-CoA, ATP, and intermediates of the TCA cycle, succinyl-CoA and alpha-ketoglutarate, behaved as inhibitors in vitro. Using pig and chicken heart enzymes for comparison, we found similarities at the N-terminal region. However, in the Ouchterlony immunodiffusion test, the polyclonal antibody raised against Drosophila citrate synthase did not show any crossreaction with pig, chicken or pigeon enzymes.
Mol Cells 1997 Oct 31
PMID:Characterization of citrate synthase purified from Drosophila melanogaster. 938 45

Enterocytes from fasted rabbits make glucose from exogenous fructose and dihydroxyacetone at rates of 180 and 91 nmol/min/10(8) cells but do not make glucose from glycerol, aspartate, malate, lactate, alpha-ketoglutarate, glutamate or glutamine. Total activities of phosphoenolpyruvate carboxykinase, fructose 1,6-bisphosphatase and glucose 6-phosphatase in isolated enterocytes are 0.44, 0.60 and 1.90 mumol/min/10(8) cells, and > or = 95% of carboxykinase activity is intramitochondrial. Enterocytes contain marginal glycerol kinase (0.05 mumol/ min/10(8) cells) and essentially no pyruvate carboxylase activities. Enterocyte mitochondria synthesize citrate from exogenous phosphoenolpyruvate and acetylcarnitine at a rate of 2.40 nmol/min/mg protein. Citrate formation is highly dependent on exogenous HCO3 and inhibited strongly by 3-mercaptopicolinate and 1,2,3-benzenetricarboxylate. Citrate synthesis is stimulated consistently by GDP and significantly so by GTP. Citrate production is unaffected by ADP or ATP. Enterocytes from fasted-refed rabbits contain activities of 0.05, 0.12, 0.39 and 0.56 mumol/min/mg cytosolic protein of ATP:citrate lyase, NADP:malate dehydrogenase, glucose 6-phosphate dehydrogenase and NADP:isocitrate dehydrogenase. Activities of NADP:malate dehydrogenase, glucose 6-phosphate dehydrogenase and NADP:isocitrate dehydrogenase are significantly higher in enterocytes from fasted-refed rabbits than those from fasted rabbits. Mitochondrial phosphoenolpyruvate carboxykinase in enterocytes in vivo could convert glycolysis-derived phosphoenolpyruvate to oxaloacetate that, with acetyl CoA, could form citrate for export to support cytosolic lipogenesis as an activator of acetyl CoA carboxylase, a source of carbon via ATP:citrate lyase and of NADPH via NADP:malate dehydrogenase or NADP:isocitrate dehydrogenase.
Comp Biochem Physiol B Biochem Mol Biol 1997 Nov
PMID:Synthesis of citrate from phosphoenolpyruvate and acetylcarnitine by mitochondria from rabbit enterocytes: implications for lipogenesis. 946 72


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