Showing papers on "Methylglyoxal published in 1993"

The formation of methylglyoxal from triose phosphates. Investigation using a specific assay for methylglyoxal.

Abstract: In Krebs-Ringer phosphate buffer, the rate of formation of methylglyoxal from glycerone phosphate and glyceraldehyde 3-phosphate was first order with respect to the triose phosphate with rates constant values of 1.94 +/- 0.02 x 10(-5) s-1 (n = 18) and 1.54 +/- 0.02 x 10(-4) s-1 (n = 18) at 37 degrees C, respectively. The rate of formation of methylglyoxal from glycerone phosphate and glyceraldehyde 3-phosphate in the presence of red blood cell lysate was not significantly different from the non-enzymatic value (P > 0.05). Methylglyoxal formation from glycerone phosphate was increased in the presence of triose phosphate isomerase but this may be due to the faster non-enzymatic formation from the glyceraldehyde 3-phosphate isomerisation product. For red blood cells in vitro, the predicted non-enzymatic rate of formation of methylglyoxal from glycerone phosphate and glyceraldehyde 3-phosphate may account for the metabolic flux through the glyoxalase system. The reactivity of glycerone phosphate and glyceraldehyde 3-phosphate towards the non-enzymatic formation of methylglyoxal under physiological conditions suggests that methylglyoxal formation is unavoidable from the Embden-Meyerhof pathway.

Mechanism for the formation of methylglyoxal from triosephosphates

Abstract: Very early in an investigation of the mechanism for the interconversion of dihydroxyacetone phosphate (DHAP) and 1)-glyceraldehyde-3-phosphate (1)GAP) by isomerization I noticed the surprising fact that the isomerization reaction could be detected only against the background of the much faster degradation of these compounds to give methylglyoxal and inorganic phosphate. DHAP and I)GAP are intermediates of glycolysis and, as such, would not be expected to undergo spontaneous degradation to methylglyoxal, a compound that inhibits cellular growth at low concentrations and that is toxic at high concentrations [ 11. Glycolytic enzymes are present at extremely high cellular concentrations to maintain the very large flux of catabolites needed to ‘stoke the cellular engine’. It is difficult to imagine how the spill-off of the toxic byproduct methylglyoxal from this pathway could not have important metabolic consequences, but these have been given relatively little consideration. This paper summarizes investigations of the mechanism for the non-enzymic and triosephosphate-isomerase-catalysed elimination reactions of triosephosphates and the conclusions from this work about the surprising kinetic instability of these compounds. It concludes with a brief commentary on the possible metabolic consequences of these elimination reactions.

Activation of potassium channels during metabolite detoxification in Escherichia coli

Abstract: In bacteria the detoxification of compounds as diverse as methylglyoxal and chlorodinitrobenzene proceeds through the formation of a glutathione adduct. In the Gram-negative bacteria, e.g. Escherichia coli, such glutathione adducts activate one, or both, of a pair of potassium efflux systems KefB and KefC. These systems share many of the properties of cation-translocating channels in eukaryotes. The activity of these systems has been found to be present in a range of Gram-negative bacteria, but not in the glutathione-deficient species of Gram-positive organisms. The conservation of the activity of these systems in a diverse range of organisms suggested a physiological role for these systems. Here we demonstrate that in E. coli cells activation of the KefB efflux system is essential for the survival of exposure to methylglyoxal. Methylglyoxal can be added to the growth medium or its synthesis can be stimulated in the cytoplasm. Under both sets of conditions survival is aided by the activity of KefB. Inhibition of KefB activity by the addition of 10 mM potassium to the growth medium stimulates methylglyoxal-induced cell death. This establishes an essential physiological function for the KefB system.

A method for the detection of carbonyl compounds in wine: Glyoxal and methylglyoxal

Abstract: Wine aldehydes were identified as O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine derivatives by GC-MS or with a GC-electron-capture detector. This method has been used to evaluate levels of glyoxal and methylglyoxal in wine. Reproducibility and linearity studies gave satisfying results. Glyoxal and methylglyoxal are formed during fermentation. Among the factors affecting their production, high musts pH increased the levels found in the corresponding wines. Various microorganisms of the wine such as Saccharomyces cerevisiae and Leuconostoc aenos can produce glyoxal and methylglyoxal. The concentrations in Sherry wines were particularly high. Because of the toxicological properties of these substances, their determination and the knowledge of their metabolism by wine microorganisms are very important.

Inhibition of glycolysis and mitochondrial respiration of Ehrlich ascites carcinoma cells by methylglyoxal.

Abstract: The effect of methylglyoxal (MG) on the aerobic glycolysis of Ehrlich ascites carcinoma (EAC) cells has been tested. Methylglyoxal inhibited glucose utilization and glucose 6-phosphate (G6P) and L-lactate formation in whole EAC cells. Methylglyoxal strongly inactivated glyceraldehyde 3-phosphate dehydrogenase (GA3PD) of the malignant cells, whereas MG has little inactivating effect on this enzyme from several normal sources. Methylglyoxal also inactivated only the participate hexominase of the EAC cells, but this inactivation was less pronounced than the effect on GA3PD. Methylglyoxal has little inactivating effect on glucose 6-phosphate dehydrogenase (G6PD), and no effect on L-lactate dehydrogenase (LDH) of the malignant cells. Glucose-dependent L-lactic acid formation of EAC-cell-free homogenate was strongly inhibited by MG, but when GA3PD of normal cells was added to this homogenate, significant lactate formation was observed even in the presence of MG. Methylglyoxal also inhibited the respiration of EAC-cell mitochondria. Respiration of mitochondria isolated from liver and kidney of normal mice, however, remained unaffected. As a consequence of the inhibition of glycolysis and mitochondrial respiration, the ATp level of the EAC cells was drastically reduced. Studies reported herein strongly suggest that the tumoricidal effect of MG is mediated at least in part through the inhibition of mitochondrial respiration and inactivation of GA3PD, and this enzyme may play an important role in the high glycolytic capacity of the malignant cells.

Purification and characterisation of glyoxalase II from human red blood cells.

Abstract: Glyoxalase II was purified from human red blood cells. The purification factor was 83 300 and the yield was 24% or 1.7 μg/ml red blood cells. The purified protein was a monomer with a molecular mass of 29 200 Da and an isoelectric point of 8.3. The rate of hydrolysis of S-D-lactoylglutathione to reduced glutathione and D-lactate, catalysed by glyoxalase II, followed Michaelis-Menten kinetics where the Km and kcat values where 146 ± 9 μM and 727 ± 16 s−1, respectively in 50 mM Tris/HCl, pH 7.4 at 37 °C. Other S-2-hydroxyacylglutathione derivatives were also acceptable substrates. S-p-Nitrobenzoxycarbonylglutathione was a potent competitive inhibitor of glyoxalase II with a Ki value of 1.20 ± 0.21 μM, and the hemithioacetal formed non-enzymically from the reaction of methylglyoxal with reduced glutathione was a weak competitive inhibitor with a Ki value of 834 ± 98 μM.

A simplified method for the purification of human red blood cell glyoxalase. I. Characteristics, immunoblotting, and inhibitor studies

Abstract: Glyoxalase I (EC 4.4.1.5) was purified from human red blood cells by a simplified method using S-hexylglutathione affinity chromatography with a modified concentration gradient of S-hexylglutathione for elution. The pure protein had a specific activity of 1830 U/mg of protein, where the overall yield was 9%. The pure protein had a molecular mass of 46,000 D, comprised of two subunits of 23,000 D each, and an isoelectric point value of 5.1. TheK M value for methylglyoxal-glutathione hemithioacetal was 192±8 µM and thek cat value was 10.9±0.2 × 104 min−1 (N = 15). The glyoxalase I inhibitor S-p-bromobenzylglutathione had aK i value of 0.16±0.04 µM and S-p-nitrobenzoxycarbonylglutathione, previously thought to inhibit only glyoxalase II, also inhibited glyoxalase I with aK i value of 3.12±0.88 µM. Reduced glutathione was a weak competitive inhibitor of glyoxalase I with aK i value of 18±8 mM. The polyclonal antibodies were raised to the purified enzyme and were found to react specifically with glyoxalase I antigen by immunoblotting. This procedure gave a protein of high purity with simple low pressure chromatographic techniques with a moderate but adequate yield for small-scale preparations.

Glucose toxicity in Prevotella ruminicola: methylglyoxal accumulation and its effect on membrane physiology.

Abstract: When the ruminal bacterium prevotella ruminicola B(1)4-M was grown in a defined medium with an excess of glucose (3.6 mM ammonia and 50 mM glucose), the cells accumulated large amounts of cellular polysaccharide and the viable cell number decreased at least 1,000-fold. This decrease in viability was correlated with an accumulation of methylglyoxal in the supernatant (3 to 4 mM). Other genetically distinct strains of P. ruminicola produced methylglyoxal, but methylglyoxal production was not ubiquitous among the strains. When P. ruminicola B(1)4-M was grown in continuous culture (dilution rate, 0.1 h-1) with an excess of glucose, there was an oscillating pattern of growth and cell death which was correlated with the accumulation and washout of methylglyoxal from the culture vessel. Mutants which resisted an excess of glucose took up glucose at a slower rate and produced less methylglyoxal than the wild type. These mutants were, however, not stable. There was always a long lag time, and the mutants could only be maintained with a daily transfer schedule. When the mutants were transferred less frequently, methylglyoxal eventually accumulated and the cultures died. The mutants transported glucose at a threefold-slower rate than the wild type, and in each case the carrier had more than one binding site for glucose. Because glucose transport could not be driven by phosphoenolpyruvate or ATP, the glucose carrier of P. ruminicola is probably a proton symport system. When P. ruminicola B(1)4-M cultures were treated with 4 mM methylglyoxal, the delta psi decreased even though intracellular ATP concentrations were high.(ABSTRACT TRUNCATED AT 250 WORDS)

Has reactive oxygen a role in methylglyoxal toxicity? A study on cultured rat hepatocytes.

Abstract: The toxicity of methylglyoxal and its ability to generate reactive oxygen species were investigated in cultured rat hepatocytes. Under aerobic and anaerobic conditions methylglyoxal increased lactate dehydrogenase (LDH) release and trypan blue uptake in a concentration dependent manner. Those concentrations of methylglyoxal causing cell injury (1 mM<) also caused the release of reactive oxygen species as indicated by peroxidase-catalyzed luminol chemiluminescence. Release of reactive oxygen was detectable only under aerobic conditions, and only became significant when a large portion of the cells had already lost their viability. It is concluded that methylglyoxal injures cultured rat hepatocytes and induces the generation of reactive oxygen species. The reactive oxygen species, however, are essentially not involved in methylglyoxal hepatotoxicity but are released by already severely injured cells.

Modification of the amino group of guanosine by methylglyoxal and other alpha-ketoaldehydes in the presence of hydrogen peroxide.

Abstract: Methylglyoxal is directly mutagenic to Salmonella typhimurium TA100 and its mutagenicity is markedly enhanced in the presence of hydrogen peroxide. We found that methylglyoxal in phosphate buffer was decomposed easily by hydrogen peroxide at room temperature to yield acetic acid and formic acid as major products and diacetyl as a minor product; acetyl radical was detected in the solution by ESR spectroscopy by the use of a spin-trapping reagent, 5,5-dimethyl-1-pyrroline N-oxide. Furthermore, guanosine was converted into N2-acetylguanosine by a combination of methylglyoxal and hydrogen peroxide in 0.1 M phosphate buffers (pH 6.1 to 7.4). This acetylation may be related to the enhancement of methylglyoxal mutagenicity by hydrogen peroxide. Other alpha-ketoaldehydes such as glyoxal and phenylglyoxal also yielded the corresponding acids and alpha-dicarbonyls upon reaction with hydrogen peroxide under the same conditions as above. These acids would have been produced through Baeyer-Villiger reaction or coupling of acyl radical with hydroxy radical, and dicarbonyls by dimerization of acyl radicals. In addition, when phenylglyoxal was used, the generation of benzoyl radical and the conversion of guanosine to N2-benzoylguanosine were observed. However, it remains to be established whether the generation of acyl radicals is directly involved in the N-2 acylation of guanosine.

Inhibition of proliferation of human leukaemia 60 cells by methylglyoxal in vitro.

Abstract: Methylglyoxal (2-oxopropanal) is the physiological substrate of the glyoxalase system. When exogenous methylglyoxal (50 μM–1 mM) was added to human leukaemia 60 (HL60) cells in culture (5 × 10 4 cells/ml), inhibition of growth and toxicity was induced. The median growth inhibitory concentration ic 50 value was 238 ± 2 μM. There was little differentiation of HL60 cells induced by methylglyoxal (a maximum of 2% differentiation with 500 μM methylglyoxal). There was no similar toxicity induced by methylglyoxal in corresponding differentiated cells, neutrophils, under the same culture conditions. Cell growth and toxicity induced by methylglyoxal (250 μM) in HL60 cells occurred in the initial 24 h of culture, after which residual surviving cells exhibited normal growth kinetics. It could also be prevented by replacing the culture medium in the initial 6 h of culture; thereafter, irreversible toxicity developed, reaching the maximum value after 24 h of culture. Growth arrest and toxicity induced by methylglyoxal increased with increasing serum composition of the medium. The mechanism of toxicity is unknown.

Genotoxicity of Environmental Mutagens in a Drosophila DNA-repair Test

Abstract: Genotoxicity of 13 mutagens was tested by a Drosophila DNA-repair test. Although strong mutagens such as aflatoxin B1, sterigmatocystin, 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide, 2-amino-3-methylimidazo [4, 5-f] quinoline, 2-amino-3, 4-dimethylimidazo [4, 5-f] quinoline, 2-amino-3, 8-dimethylimidazo [4, 5-f] quinoxaline, 3-amino-1, 4-dimethyl-5H-pyrido [4, 3-b] indole, 3-amino-1-methyl-5H-pyrido [4, 3-b] indole, and 1, 8-dinitropyrene showed positive results, methylglyoxal, formaldehyde, kojic acid, and hydrogen peroxide gave negative outcome. The ability of larval S9 fraction to activate or inactivate mutagens was monitored in the umu test. In the presence of larval S9, AFB1, IQ, Trp-P-1 and Trp-P-2 induced the umu gene expression, depending on their genotoxicities in the DNA repair test. In contrast, the genotoxicities of AF-2 and methylglyoxal in the umu test were diminished in the presence of S9 fraction. These results suggest that the genotoxic potency of a compound in the repair test is affected at least partly by metabolism in the larvae.

In Vitro Testing of a Glyoxalase I Inhibitor

Abstract: Glyoxalase I (S-D-lactoylglutathione methylglyoxal lyase (isomerising), EC 4.4.1.5) and glyoxalase I1 (S-2hydroxyacylglutathione hydrolase, EC 3.1.2.6) catalyze the conversion of toxic a-ketoaldehydes to nontoxic ahydroxycarboxylic acids. Endogenously formed methylglyoxal is converted to D-lactic acid by the tandem action of these two ubiquitous enzymes and catalytic quantities of reduced glutathione (1). The primary biological role of the glyoxalase system is not known but the enzyme system may be involved in regulation of cell division (2), threonine degradation (11, regulation of heme biosynthesis (3) and methylglyoxal detoxification (1). Recent evidence indicates that methylglyoxal is formed as a side product (0.4 mM/cell/day) during the conversion of dihydroxyacetone phosphate to glyceraldehyde 3phosphate by triosephosphate isomerase (43). Methylglyoxal is also formed enzymatically from triose phosphates (6) and from acetone by hepatic acetoneinducible cytochrome P,,, (7). Hence, the glyoxalase system serves as one of the major pathways for aketoaldehyde metabolism. Inhibitors of the glyoxalase system may prove to be useful in controlling or eliminating certain pathologies (cancer and inflammatory processes). Such inhibitors may also be useful as probes of the glyoxalase system. For example, it was recently shown that the malarial parasite Plasmodium falciparum consumes large amounts of glucose during the intraerythrocyte stage of its lifecycle and that 6-7% of the glucose consumed is converted to D-lactate via the glyoxalase system (8). Several glutathione analogues were tested against yeast glyoxalase I. Under our assay conditions (9), the compound S-(N-phenethylthiocarbamoy1)-L-glutathione (Figure 1) was determined to have an IC,, of approximately 156 pM (10). As this compound was

Kinetics of alpha-dicarbonyls reduction by L-glycol dehydrogenase (NAD+) from Enterobacter aerogenes.

Abstract: L-glycol dehydrogenase from Enterobacter aerogenes shows a high affinity by NADH (Ks = 2-4 microM; Km = 4.3-9.7 microM), which indicates that it must operate in vivo saturated with this coenzyme. Michaelis and dissociation constants for the reduction of the carbonyl substrates assayed (diacetyl, 2,3-pentanedione, methylglyoxal and ethyl pyruvate) are similar to those reported for other diacetyl reducing enzymes. The kinetic mechanism followed by these reactions has also been studied. Our results prove that the reduction of diacetyl and ethyl pyruvate takes place via an Ordered Bi-Bi system with the coenzyme as the leading substrate. Methylglyoxal and 2,3-pentanedione are reduced by the same mechanism or by a Theorell-Chance one.

Behavior of Mutagenic Aldehydes formed by Preozonation in Postchlorination.

Abstract: p‐Hydroxybenzaldehyde, one of components of humic substances, were chlorinated after preozonation. Mutagenic aldehydes, such as formaldehyde, acetaldehyde, glyoxal, glyoxylic acid and methylglyoxal were detected in residual water layer after ether extraction. With the elevation of added chlorine dose, glyoxal, glyoxylic acid and methylglyoxal decreased, but formaldehyde and acetaldehyde increased.

Glucose Toxicity inPrevotella ruminicola: Methylglyoxal Accumulation andItsEffect on MembranePhysiology

Abstract: Whentheruminal bacterium Prevotella ruminicola B14-Mwas growninadefined mediumwithan excessof glucose (3.6 mM ammoniaand50mM glucose), thecells accumulated large amountsofcellular polysaccharide andtheviable cell numberdecreased atleast 1,000-fold. Thisdecrease inviability was correlated withan accumulation ofmethylglyoxal inthesupernatant (3to4 mM).Othergenetically distinct strains ofP. ruminicola produced methylglyoxal, butmethylglyoxal production was notubiquitous amongthestrains. When P.ruminicola B14-Mwas grown incontinuous culture (dilution rate, 0.1h-1) withan excessofglucose, there was an oscillating pattern ofgrowth andcell deathwhichwas correlated withtheaccumulation andwashout ofmethylglyoxal fromtheculture vessel. Mutants whichresisted anexcessofglucose tookupglucose ataslower rateandproduced less methylglyoxal thanthewildtype. Thesemutants were,however, notstable. Therewas always a longlagtime, andthemutants could onlybemaintained witha daily transfer schedule. When the mutants were transferred less frequently, methylglyoxal eventually accumulated andthecultures died. The mutantstransported glucose atathreefold-slower ratethanthewildtype, andineach casethecarrier hadmore thanone binding site forglucose. Because glucose transport could notbedriven byphosphoenolpyruvate or ATP,theglucose carrier ofP.ruminicola isprobably a proton symport system. WhenP.ruminicola B14-M cultures were treated with4mM methylglyoxal, theA*decreased eventhough intracellular ATP concentrations were high. Thedecrease inA*was associated witha decline intheintracellular potassium level andan inhibition ofhigh-affinity glucose transport. Dicyclohexylcarbodiimide, an inhibitor oftheF1F0ATPase, had a similar effect onA*,intracellular potassium level, andhigh-affinity glucose transport. Inruminant animals, foodstuffs arefermented inthe rumenprior togastric andintestinal digestion, andthe animal mustdepend onthefermentation endproducts (volatile fatty acidsandmicrobial protein) formuchofits nutrition. Thestudy ofrumenfermentation hasbeenconfounded bythediversity ofruminal microorganisms. Enumeration studies indicated thatthenumbersofindividual bacteria varywithdiet andduring thecourseofafeeding cycle, butthese trends haveneverbeenquantitated ina systematic fashion (2,3,8,18). WhenCosterton etal.(4)examined ruminal bacteria with anelectron microscope, theynotedthat Prevotella ruminicolaaccumulated large amountsof"electron-light carbohydrate material" andthatthecytoplasm ofsomecells was "nearly filled bythis substance." Howlett etal.(7)reported that anthrone-reactive material accounted forapproximately 30%ofthedryweight ofP.ruminicola B14andthat half of thepolysaccharide waspresent asintracellular granules. The relationship between this intracellular "storage" andviabilitywas,however, notconsidered.