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PubMed1996

Effect of alpha-KG in lumen on PAH transport by isolated perfused rabbit renal proximal tubules.

To determine whether dicarboxylate taken up at the luminal membrane could function in the p-aminohippurate (PAH) countertransport at the basolateral membrane, we examined the effect of adding alpha-ketoglutarate (alpha-KG) or glutarate (a nonmetabolized dicarboxylate that is countertransported for PAH at the basolateral membrane) to the luminal perfusate on net secretion of radiolabeled PAH in isolated perfused S2 segments of rabbit proximal tubules. Addition of 100 microM alpha-KG or glutarate to the luminal perfusate in tubules perfused and bathed with HEPES-buffered medium (in the absence of bicarbonate, glycine, lactate, malate, and citrate) produced a reversible twofold stimulation of net PAH transepithelial secretion. Addition of 4 mM LiCl (an inhibitor of Na-dicarboxylate transport that does not directly affect PAH transport) to the luminal perfusate along with alpha-KG eliminated stimulation of net PAH secretion. Addition of 100 microM or 1 mM alpha-KG or glutarate to the luminal perfusate in tubules perfused and bathed with bicarbonate-buffered medium containing glycine, lactate, malate, and citrate had no effect on net PAH transport from bath to lumen. These data indicate that alpha-KG (or glutarate) that enters the tubule cells via the luminal Na-dicarboxylate cotransporter can stimulate net PAH secretion, apparently via countertransport at the basolateral membrane, but only when tubules are not in an optimal metabolic state to produce intracellular alpha-KG.

Animals↗
PubMed1996

Mechanism mediating basolateral transport of 2,4-dichlorophenoxyacetic acid in rat kidney.

The organic anions, p-aminohippurate (PAH) and fluorescein, are transported across the basolateral membrane of the renal proximal tubule in exchange for intracellular alpha-ketoglutarate (alpha KG), a mechanism indirectly coupled to sodium via Na+/alpha KG cotransport. To determine whether this mechanism mediates the basolateral transport of other organic anions, transport of the herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), was examined in rat renal cortical slices and basolateral membrane vesicles. In slices, uptake of 2,4-D increased steadily over time, approaching steady-state tissue/medium ratios of approximately 8 after 60 min. Probenecid, PAH and chlorophenol red inhibited steady-state uptake of 2,4-D. Accumulation of 10 microM 2,4-D was stimulated 2-fold by 60 microM glutarate; other dicarboxylic acids failed to stimulate uptake. In the presence of sodium, the addition of 5 mM LiCl or 2 mM ouabain to the bathing medium abolished glutarate stimulation. Removal of sodium from the bathing medium reversibly inhibited uptake as much as 75%. Furthermore, PAH inhibited 2,4-D uptake by slices in a dose-dependent manner, and increasing the external 2,4-D concentration decreased the inhibitory potency of PAH. In basolateral membrane vesicles, unlabeled 2,4-D inhibited sodium glutarate-coupled uptake of 3H-labeled PAH and 2,4-D in a concentration-dependent manner. Moreover, concentrative uptake of 2,4-D into vesicles could be driven by an outwardly directed gradient of glutarate or alpha KG that was generated by lithium-sensitive Na+/dicarboxylate cotransport or imposed experimentally. An outwardly directed gradient of unlabeled 2,4-D or PAH also stimulated uptake of 2,4-D. Based on these data, basolateral accumulation of 2,4-D by the renal proximal tubule is mediated by 2,4-D/alpha KG exchange, a mechanism energetically coupled to Na+/alpha KG cotransport and shared with PAH.

2,4-Dichlorophenoxyacetic Acid↗
PubMed2000

Metal Ion Catalysis in the Hydrolysis of Esters of 2-Hydroxy-1,10-phenanthroline: The Effects of Metal Ions on Intramolecular Carboxyl Group Participation.

Rate constants have been determined for hydrolysis of the acetate, glutarate, and phthalate monoesters of 2-hydroxy-1,10-phenanthroline in water at 30 degrees C and &mgr; = 0.1 M with KCl. The hydrolysis reactions of the esters are hydroxide ion catalyzed at pH > 9. The phthalate and glutarate monoesters have in addition pH-independent reactions from pH 5.5 to 9 that involve intramolecular participation by the neighboring carboxylate anion. The pH-independent reaction of the glutarate monoester is approximately 5-fold faster than that of the phthalate monoester. The plots of log k(obsd) vs pH for hydrolysis of the carboxyl substituted esters are bell shaped at pH < 5, which indicates a rapid reaction of the zwitterionic species (carboxyl anion and protonated phenanthroline nitrogen). The divalent metal ions, Cu(2+), Ni(2+), Zn(2+), and Co(2+), complex strongly with the esters; saturation occurs at metal ion concentrations less than 0.01 M. The 1:1 metal ion complexes have greatly enhanced rates of hydrolysis; the second-order rate constants for the OH(-) reactions are increased by factors of 10(5) to 10(8) by the metal ion. The pH-rate constant profiles for the phthalate and glutarate ester metal ion complexes have a sigmoidal region below pH 6 that can be attributed to a metal ion-promoted carboxylate anion nucleophilic reaction. The carboxyl group reactions are enhanced 10(2) - to 10(3) -fold by the metal ions, which allows the neighboring group reaction to be competitive with the favorable metal ion-promoted OH(-) reaction at pH < 6, but not at pH > 6. The half-lives of the pH-independent neighboring carboxyl group reactions of the Cu(II) complexes at 30 degrees C are approximately l2 s. The other metal ion complexes are only slightly less reactive (half-lives vary from 2.5 to 40 s). These are the most rapid neighboring carboxyl group reactions that have been observed in ester hydrolysis. Copyright 2000 Academic Press.

Journal Article↗
PubMed1986

Metabolism and toxicity of synthetic analogues of macrocyclic diester pyrrolizidine alkaloids.

Seven macrocyclic diesters analogous to hepatotoxic pyrrolizidine alkaloids have been tested in male weanling Wistar rats. The compounds were the succinate (VII), 2,3-dimethylsuccinate (VIII), phthalate (IX), glutarate (X), 2,4-dimethylglutarate (XI), 3,3-dimethylglutarate (XII) and 3,3-pentamethyleneglutarate (XIII) of the synthetic amino dialcohol, synthanecine A. Single doses of these compounds were given i.p. to rats, and liver levels of pyrrolic metabolites were measured 2 h later. For these experiments both normal rats and rats pretreated with the esterase inhibitor tri-orthocresylphosphate (TOCP) were used. In normal rats, low levels of pyrrolic metabolites were formed from compounds VII, IX, X and XI, but these levels were greatly enhanced in rats with inhibited esterase activity. Much higher pyrrole levels were formed from compounds VIII, XII and XIII in normal rats, and esterase inhibition had relatively little effect on their metabolic conversion to pyrroles. This indicated that the last mentioned compounds were relatively resistant to enzymic hydrolysis, whereas VII, IX, X and XI were easily hydrolysed in normal rats, providing an alternative metabolic path which limited their conversion to pyrrolic metabolites. Comparison of results obtained using the 2,4-dimethylglutarate (XI), the 3,3-dimethylglutarate (XII) and the 3,3,-pentamethyleneglutarate (XIII) showed that 3,3-disubstitution but not 2,4-disubstitution in the glutaric acid moiety conferred high resistance to esterase attack. Toxicity tests using four of the compounds confirmed that acute hepatotoxicity was dose related, and associated with the formation of pyrrolic metabolites in the liver. The 3,3-dimethylglutarate (XII) was highly toxic both in normal and in TOCP treated rats, doses of 25-30 mg/kg causing moderate to severe centrilobular necrosis of the liver. In contrast the toxicity of the unsubstituted succinate (VII), glutarate (X) and 2,4-dimethylglutarate (XI) was very low in normal rats but high in rats with inhibited esterase activity. Thus, the glutarate (X) was non-toxic at 200 mg/kg in normal rats, but in TOCP treated rats, in which pyrrolic metabolite formation was enhanced by a factor of 17.5, a 50 mg/kg dose of this compound was severely hepatotoxic. Kidney damage, which was generally limited to the presence of isolated necrotic cells, sometimes accompanied the liver damage caused by these compounds, but acute toxic effects were not observed in any other tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2006

Overall kinetic mechanism of saccharopine dehydrogenase from Saccharomyces cerevisiae.

Kinetic data have been measured for the histidine-tagged saccharopine dehydrogenase from Saccharomyces cerevisiae, suggesting the ordered addition of nicotinamide adenine dinucleotide (NAD) followed by saccharopine in the physiologic reaction direction. In the opposite direction, the reduced nicotinamide adenine dinucleotide (NADH) adds to the enzyme first, while there is no preference for the order of binding of alpha-ketoglutarate (alpha-Kg) and lysine. In the direction of saccharopine formation, data also suggest that, at high concentrations, lysine inhibits the reaction by binding to free enzyme. In addition, uncompetitive substrate inhibition by alpha-Kg and double inhibition by NAD and alpha-Kg suggest the existence of an abortive E:NAD:alpha-Kg complex. Product inhibition by saccharopine is uncompetitive versus NADH, suggesting a practical irreversibility of the reaction at pH 7.0 in agreement with the overall K(eq). Saccharopine is noncompetitive versus lysine or alpha-Kg, suggesting the existence of both E:NADH:saccharopine and E:NAD:saccharopine complexes. NAD is competitive versus NADH, and noncompetitive versus lysine and alpha-Kg, indicating the combination of the dinucleotides with free enzyme. Dead-end inhibition studies are also consistent with the random addition of alpha-Kg and lysine. Leucine and oxalylglycine serve as lysine and alpha-Kg dead-end analogues, respectively, and are uncompetitive against NADH and noncompetitive against alpha-Kg and lysine, respectively. Oxaloacetate (OAA), pyruvate, and glutarate behave as dead-end analogues of lysine, which suggests that the lysine-binding site has a higher affinity for keto acid analogues than does the alpha-Kg site or that dicarboxylic acids have more than one binding mode on the enzyme. In addition, OAA and glutarate also bind to free enzyme as does lysine at high concentrations. Glutarate gives S-parabolic noncompetitive inhibition versus NADH, indicating the formation of a E:(glutarate)2 complex as a result of occupying both the lysine- and alpha-Kg-binding sites. Pyruvate, a slow alternative keto acid substrate, exhibits competitive inhibition versus both lysine and alpha-Kg, suggesting the combination to the E:NADH:alpha-Kg and E:NADH:lysine enzyme forms. The equilibrium constant for the reaction has been measured at pH 7.0 as 3.9 x 10(-7) M by monitoring the change in NADH upon the addition of the enzyme. The Haldane relationship is in very good agreement with the directly measured value.

Binding Sites↗
PubMed1977

Catabolism of L-lysine by Pseudomonas aeruginosa.

Pseudomonas aeruginosa PACI grows poorly on L-lysine as sole source of carbon but mutant derivatives which grow rapidly were readily isolated. Studies with one such mutant, P. aeruginosa PAC586, supported the existence of a route for L-lysine catabolism which differes from those reported previously in other species of Pseudomonas. The postulated route, the cadaverine or decarboxylase pathway, is initiated by the decarboxylation of L-lysine and involves the following steps: L-lysine leads to cadverine leads to I-piperideine leads 5-aminovalerate leads to glutarate semialdehyde leads glutarate. Evidence for this pathway is based on the characterization of the pathway reactions and the induction of the corresponding enzymes by growth on L-lysine. The first three enzymes were also induced by growth on cadaverine and to a lesser extent by 5-aminovalerate. No evidence was obtained for the presence of pathways involving L-lysine 2-monooxygenase or L-pipecolate dehydrogenase, but another potential route for L-lysine catabolism initiated by L-lysine 6-aminotransferase was detected. Studies with mutants unable to grow on L-lysine supported the existence of more than one catabolic pathway for L-lysine in this organism and indicated that all routes converge on a pathway for glutarate catabolism which generates acetyl-CoA. Pipecolate catabolism also appeared to converge on the glutarate pathway in P. AERUGINOSA. The results suggested that the growth rate of the parental strain is limited by the rate of transport and/or decarboxylation of L-lysine. The cadaverine pathway was present, but not so highly induced, in the parental strain P. aeruginosa PACI. Pseudomonas fluorescens contained enzymes of both the cadaverine (decarboxylase) and oxygenase pathways, strains of P. putida (biotypes A and B) contained enzymes of the oxygenase pathway but not the decarboxylase pathway and P. multivorans appeared deficient in both. All these species possessed L-lysine aminotransferase activity.

Cadaverine↗
PubMed2006

Cyclohexane carboxylate and benzoate formation from crotonate in Syntrophus aciditrophicus.

The anaerobic, syntrophic bacterium Syntrophus aciditrophicus grown in pure culture produced 1.4 +/- 0.24 mol of acetate and 0.16 +/- 0.02 mol of cyclohexane carboxylate per mole of crotonate metabolized. [U-13C]crotonate was metabolized to [1,2-(13)C]acetate and [1,2,3,4,5,7-(13)C]cyclohexane carboxylate. Cultures grown with unlabeled crotonate and [13C]sodium bicarbonate formed [6-(13)C]cyclohexane carboxylate. Trimethylsilyl (TMS) derivatives of cyclohexane carboxylate, cyclohex-1-ene carboxylate, benzoate, pimelate, glutarate, 3-hydroxybutyrate, and acetoacetate were detected as intermediates by comparison of retention times and mass spectral profiles to authentic standards. With [U-(13)C]crotonate, the m/z-15 ion of TMS-derivatized glutarate, 3-hydroxybutyrate, and acetoacetate each increased by +4 mass units, and the m/z-15 ion of TMS-derivatized pimelate, cyclohex-1-ene carboxylate, benzoate, and cyclohexane carboxylate each increased by +6 mass units. With [13C]sodium bicarbonate and unlabeled crotonate, the m/z-15 ion of TMS derivatives of glutarate, pimelate, cyclohex-1-ene carboxylate, benzoate, and cyclohexane carboxylate each increased by +1 mass unit, suggesting that carboxylation occurred after the synthesis of a four-carbon intermediate. With [1,2-(13)C]acetate and unlabeled crotonate, the m/z-15 ion of TMS-derivatized 3-hydroxybutyrate, acetoacetate, and glutarate each increased by +0, +2, and +4 mass units, respectively, and the m/z-15 ion of TMS-derivatized pimelate, cyclohex-1-ene carboxylate, benzoate, cyclohexane carboxylate, and 2-hydroxycyclohexane carboxylate each increased by +0, +2, +4, and +6 mass units. The data are consistent with a pathway for cyclohexane carboxylate formation involving the condensation of two-carbon units derived from crotonate degradation with CO2 addition, rather than the use of the intact four-carbon skeleton of crotonate.

Benzoates↗
PubMed1979

Ethylmalonic-adipic aciduria. In vivo and in vitro studies indicating deficiency of activities of multiple acyl-CoA dehydrogenases.

The mechanisms underlying ethylmalonic-adipic aciduria were studied in a 5-yr-old girl. Oxidation of radioactive substrates by cultured skin fibroblasts from the proband and asymptomatic family members was also determined and compared to that by normal fibroblasts and that by cells from a patient with glutaric aciduria type II. Feeding medium-chain triglycerides promptly induced vomiting and lethargy accompanied by a pronounced increase of urinary ethylmalonate. Significant increases of serum isovalerate and urinary isovalerylglycine were observed after leucine feeding, but urinary glutarate increased only slightly after lysine feeding. Thus, the results from clinical investigation remained equivocal as to whether pathways other than fatty acid oxidation were blocked in our patient. Oxidation of [1-(14)C]butyrate by cultured skin fibroblasts from the proband was reduced to 14% of control. In vitro oxidation of [2-(14)C]lysine and [2-(14)C]leucine was also reduced to 28 and 23% of control, respectively. Much more severe reduction in oxidation of these three substrates (3, 9, and 9%, respectively) was observed in glutaric aciduria type II cells. These results indicated that in the proband, degradative pathways of fatty acids, lysine, and leucine are blocked at the steps of butyryl-CoA, glutaryl-CoA, and isovaleryl-CoA dehydrogenases, respectively, as in the case of glutaric aciduria type II. Because activities of multiple acyl-CoA dehydrogenases are reduced, a deficiency of electron-transferring flavoprotein, which serves as a hydrogen-acceptor for these dehydrogenases, is postulated as the underlying mechanisms of these two diseases, but a genetic heterogeneity was indicated by significant differences in the residual activities in these two types of cells. The hypothesis of more than one mutant allele of an autosomal recessive gene was also suggested by the study on cells from asymptomatic members of the family.

Acidosis↗
PubMed1989

[The influence of disinfectants on colloidal impression materials and dental stones].

In view of prophylaxis against hospital infections, following tests were performed: Two kinds of alginate impression materials (ALGIACE and Aligix-S) and two kinds of agar impression materials (CARTRIILLOID and RUBBERLLOID) were immersed for 30 minutes in the following six kinds of disinfectants; 0.1% benzethonium chloride (benzethonium), 1% chlorhexidine gluconate (hexidine), 1% chlorhexidine gluconate added with surfactants (hexidine surfactant), 1% sodium hypochlorite (hypochlorous acid), 2% glutaraldehyde (glutaral) and 1% formaldehyde (formalin). Changes in size of the impression materials with time were then measured. Three kinds of dental stones (SHIMOMURA DENTAL PLASTER, NEW PLASTONE and SURSTONE) were infused into the impression surfaces after treatment with disinfectants, and the surface roughness and hardness of the yielded surfaces of the samples were measured. The results obtained are as follows: 1) As to ALGIACE, although the rates of changes in size lay within the range of +/- 0.3% when immersed in hexidine, hexidine surfactant and hypochlorous acid, the infusion of either one of SHIMOMURA DENTAL PLASTER, NEW PLASTONE or SURSTONE into the impression showed an effect on the stone surface. 2) As to Algix-S, the rate of changes in size lay within the range of +/- 0.3% when immersed in all the disinfectants, and the influsions of NEW PLASTONE and SURSTONE after immersing in benzethonium, hexidine surfactant and glutaral gave a slight effect on the stone surface. 3) As to CARTRILLOID, the rates of change in size lay within the range of +/- 0.3% except for the immersion in hexidine; the influsion of SHIMOMURA DENTAL PLASTER into the impression, the influsion of NEW PLASTONE after immersing in benzethonium and formalin and the influsions of NEW PLASTONE and SURSTONE after immersing in glutaral gave a slight effect on teh stone surface. 4) As to RUBBERLLOID, the rates of changes in size lay within the range of +/- 0.2% when immersed each disinfectant; the influsion of SHIMOMURA DENTAL PLASTER after immersing in hypochlorous acid and that of either one of SHIMOMURA DENTAL PLASTER, NEW PLASTONE or SURSTONE after immersing in benzethonium, glutaral and formalin gave a slight effect on the stone surface.

Calcium Sulfate↗
PubMed1983

[Second set method for determining the immunogenicity of heart valves].

The "second set" method was used on inbred rats to study immunogenicity of the heart valves treated with a proteolytic enzyme and glutaric aldehyde and to compare it with immunogenicity of the valves treated with glutaric aldehyde alone according to Hancock's method. The valves treated by the enzyme and 0.2-0.5% glutaric aldehyde did not lead to the body sensitization in contrast to the valves exposed to 0.5% glutaric aldehyde alone. During transplantation of the latter ones, there were signs of the immunologic response on the part of the recipients and calcification of valvular tissue 70 days after subcutaneous implantation. It is assumed that pretreatment with the enzyme makes it possible to appreciably reduce immunogenicity of the heart valves.

Amylases↗
PubMed1993

[Metabolism of alpha-ketoacids in erythromycin biosynthesis in various strains of Saccharopolyspora erythraea].

The absence of pyruvate and insignificant levels of alpha-keto-glutarate in the mycelium during the fermentation cycle were characteristic of a highly active erythromycin-producing strain of Saccharopolyspora erythraea. Alpha-keto-glutarate partially excreted to the fermentoffon broth. The activity of pyruvate decarboxylase and alpha-keto-glutarate decarboxylase was detected in the cells during the entire period of the cultivation. The same regularities were observed in the chloramphenicol resistant mutant of the strain. The mycelium of a low productive strain of S.erythraea contained not only alpha-keto-glutarate but also pyruvate and excreted large amounts of keto-acids. By the activity levels of the decarboxylases the low productive strain did not differ from the highly productive one. Propanol did not influence the growth of the low productive strain and the synthesis of erythromycin by it. However, it stimulated accumulation of keto-acids and especially pyruvate in both the mycelium and fermentation broth. Relation between the intensity of keto-acid metabolism and erythromycin biosynthesis is discussed.

Carboxy-Lyases↗
PubMed2005

Conformations, energies, and intramolecular hydrogen bonds in dicarboxylic acids: implications for the design of synthetic dicarboxylic acid receptors.

The various conformers of the dicarboxylic acids HO2C--(CH2)n--CO2H, n = 1-4, were obtained using density functional methods (DFT), both in the gas phase and in the aqueous phase using a polarized continuum model (PCM). Several new conformers were identified, particularly for the two larger molecules glutaric (n = 3) and adipic acid (n =4). The PCM results show that the stability of most conformers were affected, many becoming unstable in the aqueous phase; and the energy ordering of conformers is also different. The results suggest that conformational preferences could be important in determining the design and stability of appropriate synthetic receptors for glutaric and adipic acid. Geometry changes between gas and aqueous phases were most marked in those conformers containing an intramolecular hydrogen bond. Additional calculations have probed the strength of intramolecular hydrogen bonds in these dicarboxylic acids. In the cases of glutaric and adipic acid, the strength of the intramolecular hydrogen bond were estimated to be around 28-29 kJ/mol, without any vibrational energy correction. The intramolecular hydrogen bond energies in malonic and succinic acid were also estimated from the calculated H-bond distances using an empirical relationship. Intramolecular H-bond redshifts of 170-250 cm(-1) have been estimated from the results of the harmonic frequency analyses.

Adipates↗
PubMed1999

Glutaryl-CoA dehydrogenase deficiency presenting as 3-hydroxyglutaric aciduria.

Two siblings who were found to have deficiency of glutaryl-CoA dehydrogenase were identified by the presence of large amounts of 3-hydroxyglutaric acid in the urine. Patients with this disease, termed glutaric acidemia or glutaric acidemia Type I, usually present with large amounts of glutaric acid in the urine, and amounts of 3-hydroxyglutaric acid found are less. Patients were ataxic and dystonic. Intelligence was normal. 3-Hydroxyglutaric acid in the urine was quantified by organic acid analysis via gas chromatography mass spectrometry (GCMS) and by stable isotope-dilution (internal standard) GCMS. Glutaryl-CoA dehydrogenase activity in cultured fibroblasts was found to be 2% of the control level. The nature of the mutations was identified, and both patients were found to be compound heterozygotes for R227P, which changed an arginine to a proline, and E365K, which changed a glutamate to a lysine.

Amino Acid Metabolism, Inborn Errors↗
PubMed1995

1H and 2H NMR spectroscopic studies on the metabolism and biochemical effects of 2-bromoethanamine in the rat.

Male Fischer 344 rats were dosed with 2-bromoethanamine hydrobromide (BEA, N = 6) or [1,2,2,-2H4]-bromoethanamine hydrobromide (BEA-d4, N = 6) at 150 mg/kg i.p. and urine was collected -24 to 0 hr pre-dose and at 0-2 hr, 2-4 hr, 4-8 hr and 8-12 hr post-dose (p.d.). Urine samples were analysed directly using 500 and 600 MHz 1H NMR and 92.1 MHz 2H NMR spectroscopy. The major observed effect of BEA treatment was the induction of transient elevations in urinary glutaric acid (GTA) and adipic acid (ADA) excretion lasting up to 24 hr p.d. Most of the GTA was excreted in the 0-8 hr p.d. with maximal rates of 100-120 microM/hr for each rat occurring between 4 and 8 hr p.d. in animals treated with BEA or BEA-d4. GTA and ADA were shown to be of endogenous origin as there was no detectable incorporation of the 2H label into either compound following treatment of rats with BEA-d4. Following BEA-treatment there was an initial decrease in the levels of urinary citrate, succinate, 2-oxoglutarate and trimethylamine-N-oxide. A subsequent recovery of citrate and succinate was noted following the onset of medullary nephropathy. The abnormal urinary metabolite profiles were similar to that observed in the urine of humans with glutaric aciduria type II (an inborn error of metabolism) caused by a lack of mitochondrial fatty acyl coenzyme A dehydrogenases indicating that BEA or its metabolites have similar metabolic consequences. The BEA metabolite aziridine was detected by 1H and 2H NMR spectroscopy of the urine 8 hr p.d. together with BEA itself and two novel metabolites 2-oxazolidone (OX) and 5-hydroxy-2-oxazolidone (HOX). The formation of OX requires the reaction of BEA with endogenous bicarbonate followed by a cyclisation reaction eliminating HBr. Dosing rats with authentic OX resulted in the excretion of HOX but did not cause glutaric or adipic aciduria indicating that either aziridine or BEA itself was responsible for the presumed defect in mitochondrial metabolism.

Adipates↗
PubMed1988

Fluorescence stopped-flow studies on the binding of 1,N6-etheno-NAD to bovine liver glutamate dehydrogenase.

Fluorescence stopped-flow techniques have been used to investigate the binding of the oxidised coenzyme eNAD to bovine liver glutamate dehydrogenase (L-glutamate:NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3) saturated with glutarate, a substrate analogue, by following the transient kinetics of fluorescence intensity changes associated with changes in the binding of 1,N6-etheno-NAD (eNAD) to the enzyme, using displacement by NAD, NADP, ADP or GTP. Computer simulations of the various kinetic models provide a detailed picture of the molecular interactions between the active site (site I) and regulatory sites (sites II and III), specific for adenine and guanine nucleotides, respectively. The observed enhancement of the eNAD dissociation rate constant from site I can satisfactorily be accounted for as being due to the effect of ADP or NAD (and to a lesser extent NADP) binding to site II. This provides a mechanism for the allosteric activation of this enzyme via a predominantly intrasubunit interaction. By contrast the isomerisation of the enzyme induced by ADP alone is markedly slowed down by the occupancy of site I by eNAD in the presence of glutarate. The inhibitory effect of the allosteric effector GTP correlates with a tightening of eNAD binding, causing a decrease of the coenzyme dissociation rate constant followed by a slow isomerisation of the enzyme complexed with eNAD and glutarate.

Adenosine Diphosphate↗
PubMed2006

Minute changes in composition of polymer substrates produce amplified differences in cell adhesion and motility via optimal ligand conditioning.

We explored the interplay between substratum chemistry of polymeric materials and surface-adsorbed ligand concentration (human plasma fibronectin) in the control of cell adhesion and cell motility. We found that small changes in the chemical composition of a polymeric substratum had different effects on cellular motility--depending on the concentration of preadsorbed fibronectin. We used two tyrosine-derived polyarylates, poly(DTD diglycolate) and poly(DTD glutarate), as substrata for the seeding of NIH-3T3 fibroblasts. The only compositional difference between the two test polymers was that one single oxygen atom in the polymer backbone of poly(DTD diglycolate) had been substituted by a methylene group in the backbone of poly(DTD glutarate), The two polymers had closely matched hydrophobicity and physical properties. Flat, spin-coated surfaces of these polymers were pretreated with different concentrations of human plasma fibronectin (0-20 microg/ml). After seeding with NIH-3T3 fibroblasts, we examined the adhesion and motility behavior of these cells. We found that NIH-3T3 fibroblasts migrated significantly faster on poly(DTD diglycolate), but only when the polymer surfaces were pretreated with intermediate concentrations of fibronectin. Only at these intermediate levels of ligand conditioning, did the presence of an extra oxygen atom in the backbone of poly(DTD diglycolate) relative to poly(DTD glutarate) (i) alter the overall organization/concentration of the fibronectin; (ii) weaken cell attachment strength and inhibited excessive cell spreading; and (iii) promote cell motility kinetics. These findings indicate that the biological effect of minute changes in substratum chemistry is critically dependent on the level of surface-adsorbed cell-binding ligands.

Adsorption↗
PubMed2003

Regulation of the mitochondrial ATP-synthase in health and disease.

In most tissues the mitochondrial ATP-synthase plays a central role by synthesizing the bulk of ATP. According to the classical theory of respiratory control, flux through this enzyme is solely determined by substrate (ADP) concentration while the enzyme has a fixed capacity. However, in different cell types such as rat cardiomyocytes and neurons, dog heart, human fibroblasts, and skeletal and heart muscle from children, it has been shown that active regulation of the mitochondrial ATP-synthase in response to cellular energy demand exists. For example, in rat cardiomyocytes the mitochondrial ATP-synthase activity is down-regulated in response to anoxia or mitochondrial uncoupling. By this mechanism cellular ATP is conserved, as under these conditions the ATP-synthase would work in reverse and hydrolyze ATP. When cardiomyocytes are stimulated to contract, ATP-synthase activity is up-regulated in line with the increased energy demand. Preincubation of the cardiomyocytes with positive inotropic substances results in further up-regulation of the ATP-synthase. By blocking calcium transport, it has been shown that the up-regulation of the enzyme is calcium-dependent. On a molecular level, up-regulation is probably mediated by the calcium-binding inhibitor protein (CaBI) and down-regulation via the inhibitor protein IF(1). The ATP-synthase system is disturbed under several pathophysiological conditions. First, mutations can cause a primary defect in the mitochondrial ATP-synthase (respiratory chain defect). Furthermore, secondary defects of the ATP-synthase occur. In rat models abnormalities of ATP-synthase can be detected in different types of cardiomyopathy/heart hypertrophy. The changes are reversible in response to treatment of the heart diseases. Abnormalities of the ATP-synthase system can be observed in fibroblasts from patients with neuronal ceroidlipofuscinoses. Toxic metabolites accumulating in methylmalonic acidurias can inhibit ATP-synthase. When neurons are incubated with 3-OH glutarate - a substance accumulating in glutaric aciduria I-as a model for glutaric aciduria I, ATP-synthase activity is compromised. This lack of energy may lead to 'slow onset' excitotoxicity and finally cell death. Cells can be rescued by adding creatine to the incubation medium. In D-2-hydroxyglutaric aciduria, inhibition of the ATP-synthase has been observed.

Animals↗
PubMed2001

Allosteric behaviour of 1:5 hybrids of mutant subunits of Clostridium symbiosum glutamate dehydrogenase differing in their amino acid specificity.

Hybrid hexamers were made by refolding mixtures of two mutant forms of clostridial glutamate dehydrogenase. Mutant Cys320Ser (C320S) has a similar activity to the wild-type enzyme, but is unreactive with Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoate) (DTNB). The triple mutant Lys89Leu/Ala163Gly/Ser380Ala (K89L/A163G/S380A), active with norleucine but not glutamate, is inactivated by DTNB, since the amino acid residue at position 320 is a cysteine residue. The chosen ratio favoured 1:5 hybrids of the triple mutant and C320S. The renatured mixture was treated with DTNB and separated on an NAD(+)-agarose column to which only C320S subunits bind tightly. Fractions were monitored for glutamate and norleucine activity and for releasable thionitrobenzoate to establish subunit stoichiometry. A fraction highly enriched in the 1:5 hybrid was identified. Homohexamers (C320S with 40 mM glutamate and 1 mM NAD(+) at pH 8.8, or K89L/A163G/S380A with 70 mM norleucine and 1 mM NAD(+) at pH 8.5) showed allosteric activation; succinate activated C320S approx. 50-fold (EC(50)=70 mM, h=2.4), and glutarate gave approx. 30-fold activation (EC(50)=35 mM, h=2.3). For the triple mutant, corresponding values were 80 mM and 2.2 for succinate, and 75 mM and 1.7 for glutarate, but maximal activation was only about 2-fold. In the 1:5 hybrid, with only one norleucine-active subunit per hexamer, responses to glutarate and succinate were still co-operative, and activation was more extensive than in the triple mutant homohexamer. A single norleucine-active subunit can thus respond co-operatively to a substrate analogue at the other five inactive sites. On the other hand, similar hyperbolic dependence on the norleucine concentration for the hybrid and the triple mutant homohexamer reflected the inability of C320S subunits to bind norleucine. With glutamate at pH 8.8, an h value of 3.6 was obtained for the 1:5 hybrid, in contrast with an h value of 5.2 for the C320S homohexamer. The "foreign" subunit evidently impedes inter-subunit communication to some extent.

Chromatography, Affinity↗