Showing papers on "Methylglyoxal published in 1981"

Synergistic action of two polyamine antimetabolites leads to a rapid therapeutic response in childhood leukemia.

Abstract: Sequential administration of alpha-difluoromethyl ornithine and methylglyoxal bis(guanylhydrazone), two differently acting inhibitors of the biosynthesis of natural polyamines, produced a rapid and distinct therapeutic response in four children with advanced lymphoblastic and in one with myeloblastic leukemia. The synergism between the action of the two compounds was based upon a unique drug interaction; a preceding treatment with difluoromethyl ornithine greatly increased the uptake of subsequently administered methylglyoxal bis(guanylhydrazone) as verified by the actual determinations of the latter drug in the circulating leukemia cells. The side-effects associated with the combined drug regiment were either absent or mild.

[39] Glyoxalase I (rat liver)

Abstract: Publisher Summary This chapter presents a procedure for the preparation of glyoxalase I that catalyzes the formation of S -2-hydroxyacylglutathione from a corresponding 2-oxoaldehyde and glutathione (GSH). A second enzyme, glyoxalase II, regenerates GSH by hydrolyzing the resulting thiolester. It is found that the formation of S -lactoylglutathione from methylglyoxal and GSH can be followed spectrophotometrically at 240 nm for analysis. Purification procedure are performed at about 5°, including the preparation of cytosol fraction; washing on sephadex G-25, carboxymethyl-cellulose, diethylaminoethyl cellulose column respectively; affinity chromatography with S-p -bromobenzylglutathione-sepharose 6B; washing on sephadex G-100; again affinity chromatography with S -p-bromobenzylglutathione- sepharose 6B; and chromatography on diethylaminoethyl-sepharose . The purified glyoxalase I is homogeneous in several analytical electrophoretic and chromatographic systems. In the presence of SDS, it dissociates into subunits of equal weight. Some of the known properties of glyoxalase I from the rat liver are presented in the chapter. Antibodies raised against the enzyme do not give precipitin lines with purified glyoxalase I from porcine erythrocytes, human erythrocytes, or Saccharomyces cerevisiae in Ouchterlony double-diffusion experiments.

Concentration of activated intermediates of the fructose-1,6-bisphosphate aldolase and triosephosphate isomerase reactions

Abstract: As discovered by Grazi & Trombetta [Grazi, E., & Trombetta, G. (1978) Biochem. J. 175, 361], fructose-1,6-bisphosphate aldolase of rabbit muscle causes the slow formation of inorganic phosphate (Pi) and methylglyoxal when incubated with dihydroxyacetone phosphate (DHAP). In addition, these authors found an acid-labile intermediate in equilibrium with the aldolase-dihydroxyacetone phosphate complexes representing approximately 60% of the enzyme-bound DHAP species. Experiments are reported here which argue that this acid-labile species is the enzyme-bound enamine phosphate or its equivalent that decomposes by beta elimination in acid. A similar mechanism involving an enediol phosphate is proposed to explain a phosphatase action of triosephosphate isomerase that produces methylglyoxal and Pi at the rate of approximately 0.1 s(-1) at pH 5.5. When DHAP with excess isomerase is quenched in strong acid, the formation of Pi indicates that approximately 5% of bound reactant is in the form of enediol phosphate. The remainder of the substrate is about equally distributed between bound forms of DHAP and D-glyceraldehyde 3-phosphate. This equilibrium differs by 300-fold from the appropriate equilibrium in solution. Yeast aldolase, contrary to expectation, does not catalyze formation of inorganic phosphate and methylglyoxal when incubated with DHAP and gives no evidence fro an enediol phosphate intermediate when quenched in acid.

Death of Tumor Cells in Response to the Use of a System of Stimulated Polyamine Uptake for the Transport of Methylglyoxal Bis(guanylhydrazone)

Abstract: Putrescine and spermidine depletion produced by α-difluoromethylornithine, an irreversible inhibitor of omithine decarboxylase (EC 4.1.1.17) greatly enhances the uptake of the anti-cancer drug methylglyoxal bis(guanylhydrazone) by cultured Ehrlich ascites carcinoma cells. The drug was also more effectively retained in the cells previously exposed to the inhibitor of ornithine decarboxylase. When untreated Ehrlich ascites carcinoma cells were shortly exposed to methylglyoxal bis(guanylhydrazone) and then transferred into drug-free medium, they rapidly reinitiated growth, while polyamine-deprived cells were irreversibly damaged upon similar exposure to the drug. Tumor cells with greatly reduced intracellular pools of putrescine and spermidine so effectively concentrated methylglyoxal bis(guanylhydrazone) that the drug largely disappeared from the medium resulting in a formation of concentration gradient of more than 5000-fold across the cell membrane. The tumor cells tolerated high intracellular concentrations (6-9 mM) of methylglyoxal bis(guanylhydrazone) for not more than 24 h whereafter they simply disintegrated and excreted the drug back into the medium. The final destruction of the polyaminedepleted cells in response to methylglyoxal bis(guanylhydrazone) was preceded by profound inhibition of protein synthesis, which occurred before any disturbances in thymidine incorporation were obvious. The inhibition of protein synthesis by methylglyoxal bis(guanylhydrazone) was more pronounced in cells previously exposed to difluoromethylornithine than in cells with normal intracellular polyamine pools. The extent of polyamine depletion produced by difluoromethylornithine and the rate of the uptake of methylglyoxal bis(guanylhydrazone) were positively correlated to the growth rate of the tumor cells, i.e. cells dividing slowly were more resistant towards the action of difluoromethylornithine, as regards the development of intracellular polyamine depletion, and they also accumulated methylglyoxal bis(guanylhydrazone) less effectively than did rapidly dividing cells. The rate of methylglyoxal bis(guanylhydrazone) transport was likewise related to the extent of intracellular putrescine and spermidine deprivation: tumor cells with greatly reduced putrescine and spermidine pools concentrated methylglyoxal bis(guanylhydrazone) more rapidly than cells containing only moderately decreased concentrations of the polyamines. Although the uptake of exogenous spermidine by polyamine-deprived tumor cells was initially at least as rapid as that of methylglyoxal bis(guanylhydrazone), the transport of the natural polyamine was automatically halted when the intracellular spermidine pool was brought to a normal level. It thus appears that the uptake of methylglyoxal bis(guanylhydrazone) by the stimulated transport system of polyamines leads to a suicidal accumulation of the drug and to rapid cell death.

Probing the active site of glyoxalase I from human erythrocytes by use of the strong reversible inhibitor S-p-bromobenzylglutathione and metal substitutions.

Abstract: Glyoxalase I from human erythrocytes was studied by use of the strong reversible competitive inhibitor S-p-bromobenzylglutathione. Replacements of cobalt, manganese and magnesium for the essential zinc in the enzyme were made by a new procedure involving 10% methanol as a stabilizer of the enzyme. The Km value for the adduct of methylglyoxal and glutathione was essentially unchanged by the metal substitutions, whereas the inhibition constant for S-p-bromobenzylglutathione increased from 0.08μm for the Zn-containing enzyme to 1.3, 1.7 and 2.4μm for Co-, Mn- and Mg-glyoxalase I respectively. Binding of the inhibitor to the enzyme caused quenching of the tryptophan fluorescence of the protein, from which the binding parameters could be determined by the use of non-linear regression analysis. The highest dissociation constant was obtained for apoenzyme (6.9μm). The identity of the corresponding kinetic and binding parameters of the native enzyme and the Zn2+-re-activated apoenzyme and the clear differences from the parameters of the other metal-substituted enzyme forms give strong support to the previous identification of zinc as the natural metal cofactor of glyoxalase I. Binding to apoenzyme was also shown by the use of S-p-bromobenzylglutathione as a ligand in affinity chromatography and as a protector in chemical modification experiments. The tryptophan-modifying reagent 2-hydroxy-5-nitrobenzyl bromide caused up to 85% inactivation of the enzyme. After blocking of the thiol groups (about 8 per enzyme molecule) 6.1 2-hydroxy-5-nitrobenzyl groups were incorporated. Inclusion of S-p-bromobenzylglutathione with the modifying reagent preserved the catalytic activity of the enzyme completely and decreased the number of modified residues to 4.4 per enzyme molecule. The findings indicate the presence of one tryptophan residue in the active centre of each of the two subunits of the enzyme. Thiol groups appear not to be essential for catalytic activity. The presence of at least two categories of tryptophan residues in the protein was also shown by quenching of the fluorescence by KI.

Some properties of the polyamine deprivation-inducible uptake system for methylglyoxal bis(guanylhydrazone) in tumor cells.

Abstract: Intracellular polyamine deprivation, produced by DL-alpha-difluoromethylornithine (DFMO), resulted in a striking enhancement of cellular transport of the natural polyamines and methylglyoxal bis(guanylhydrazone) (MGBG). In addition to the natural polyamines and MGBG, the uptake of other diamines and triamines was likewise enhanced in response to DFMO, although longer than three-carbon backbone was required for about 10-fold stimulation to occur. Intracellular deprivation of polyamines did not increase the affinity of the transport system for MGBG but greatly enhanced the maximum velocity of the drug transport. The uptake process of MGBG was temperature dependent and the activation energy (Ea = 67.5 kJ) for the uptake system was the same for both the polyamine-depleted tumour cells and for the untreated cells. The uptake of the drug appeared to be more dependent on the Na+-linked uptake, as indicated by the inhibitory effect of ouabain, than on energy production. Deprivation of putrescine and spermidine changed the intracellular distribution of MGBG since a major portion of the drug was concentrated in the microsomal fraction in polyamine-depleted cells.

Studies on the inactivation of glyceraldehyde-3-phosphate dehydrogenase by methylglyoxal.

Abstract: Glyceraldehyde-3-P dehydrogenase (E.C. 1.2.1.12) from rabbit muscle is rapidly inactivated by methylglyoxal following pseudo first-order kinetics. Substrate, as well as inorganic phosphate, affords protection, whereas NAD+ is ineffective. The arginine residue implicated in the reaction is probably the anion binding site of the phosphate group of the substrate.

Liberation of the triosephosphate isomerase reaction intermediate and its trapping by isomerase, yeast aldolase, and methylglyoxal synthase.

Abstract: When a mixture of triosephosphate isomerase (rabbit muscle) and dihydroxyacetone phosphate (DHAP) is quenched with acid, a compound is liberated, presumed to be the cis-enediol 3-phosphate, that decomposes to inorganic phosphate (Pi) and methylglyoxal [Iyengar, R., & Rose, I.A. (1981) Biochemistry (preceding paper is this issue)]. The decomposition can be prevented by rapid neutralization if a catalytic amount of fresh isomerase is present. Varying the time between acidification and rescue gave a half-life of the liberate compound of approximately 12-17 ms. Varying the concentration of enzyme used for rescue gave a minimum second-order rate constant for trapping of 10(9)M(-1)s(-1). These results add further evidence favoring a stepwise mechanism for the aldose-ketose isomerase reactions in which a chemically defined enzyme-bound intermediate is found. The high rate of trapping over a wide pH range indicates that the enediol phosphate, not the enediolate phosphate, is the intermediate. One property of the enzyme is to stabilize the intermediate with respect to its fragmentation in solution by greater than 1000-fold. Yeast aldolase is also able to rescue all of the isomerase intermediate, though higher concentrations of enzyme are required. Although different enantiotopic protons of DHAP are abstracted by isomerase and aldolase, both enzymes use the same enediol phosphate intermediate. Methylglyoxal synthase at a 50-fold greater concentration was unable to compete with triosephosphate isomerase for cis-enediol phosphate. Either the synthetase has a low V/K for the cis isomer or it uses the trans-enediol phosphate form specifically. A new strategy for the chemical and enzymological characterization of enzyme reaction intermediates is proved here based on the liberation of the intermediate from the reaction equilibrium and its recovery by fresh enzyme or another enzyme species.

Preparation of lactic acid and*or its ester

Abstract: PURPOSE:To obtain the titled compound useful as medicines, etc., at a low cost, by the reaction of methylglyoxal and/or its acetal with water and/or an alcohol, in the presence of a compound of a specific metal selected from groups IIIb, IVa, Va, etc. in the periodic table, as a catalyst. CONSTITUTION:Methylglyoxal and/or its acetal is reacted with water and/or an alcohol, preferably at 40 deg.C or above and 300 deg.C or below, in the presence of one or more compounds of a metal selected from groups IIIb, IVa, IVb, Va, VIb, and VII in the periodic table, e.g. Al, In, Tl, Ti, Zr, Sn, Nb, or Cr, as a catalyst in an amount of 0.05wt% or more, preferably 0.1wt% or more based on the methylglyoxal and/or its acetal, to give the titled compound.

An electron spin resonance investigation of amine models for the protein–methylglyoxal interaction

Abstract: The dicarbonyl methylglyoxal reacts with the lysine residues of proteins to give brown colored products with a small oxygen-dependent free radical content. Electron spin resonance (ESR) experiments employing ethylene diamine as a model for protein lysine residues are reported. The prevention of imine formation between methylglyoxal and the amine models either by pretreatment of methylglyoxal with glutathione or by methylation of the amine led to brightly colored reaction products and a much higher radical content. From an analysis of the ESR spectra it is clear that the reactions between methylglyoxal and the simple model amines developed far beyond those in the protein-methylglyoxal systems. It is proposed that this difference is accounted for by the enormous steric effects of the proteins. The relevance of the observations to the “browning” reaction in foods is briefly discussed.

Preparation of 4-methylimidazoles

Abstract: A process for the preparation of 4-methylimidazoles by reacting methylglyoxal with ammonia and an aldehyde in aqueous solution, wherein the reaction is carried out at a pH of above 7 and either the reactants are all brought into contact simultaneously or the aldehyde and the methylglyoxal are added simultaneously to the aqueous ammonia solution.

Production of glutathion

Abstract: PURPOSE:A methylglyoxal-resistant strain belonging to Escherichia coli and being capable of producing glutathion is cultured in a medium to accumulate glutathion, which is collected in high yield. CONSTITUTION:An above-cited methylglyoxal-resistant strain, e.g., a methylglyoxal-resistant strain (FERM-5279) derived from Escherichia coli B wild strain (ATCC 23226), is cultured in a medium containing 0.2-10% of glucose as a carbon source and 0.5-1% of ammonium sulfate as a nitrogen source. Methylglyoxal is added to the medium by about 0.7-0.4mM and the methylglyoxal resistant strain is inoculated and cultured at 25-35 deg.C and 6-7.5pH under aerobic conditions to accumulate glutathion. After cultivation, the bacterial cells are removed from the culture medium by filtration or the like and the filtrate is subjected to a well-known separation process such as treatment with ionexchange resin to collect glutathion.

Process for producing 4-methyl imidazoles

Abstract: Verfahren zur Herstellung von 4-Methylimidazolen durch Umsetzung von Methylglyoxal mit Ammoniak und Aldehyden in wasriger Losung, wobei man die Umsetzung bei einem pH-Wert von groser als 7 so durchfuhrt, das man die Komponenten gleichzeitig miteinander in Beruhrung bringt oder den Aldehyd gleichzeitig mit dem Methylglyoxal zur vorgelegten wasrigen Ammoniak-Losung gibt. A process for the preparation of 4-Methylimidazolen by reacting methylglyoxal with ammonia and aldehydes in aqueous solution, the reaction being carried out at a pH value of greater than 7 so carried out, by bringing the components simultaneously contact each other or to the aldehyde simultaneously with the are methylglyoxal to submitted aqueous ammonia solution.

Insecticide intermediate methyl:glyoxal di:methyl acetal prodn. - by reaction of aq. methyl:glyoxal with methanol in the presence of acidic catalyst with azeotropic removal of water

Abstract: Prodn. of methylglyoxal dimethyl acetal (I) from MeOH and an aq. methylglyoxal soln. in the presence of an acidic catalyst with azeotropic removal of water, is claimed where (A) in a first step, before the reaction with MeOH, the aq. methylglyoxal soln. is dried to a residual water content of 10-35 wt.% with the aid of an azeotroping agent and (B) in a second step MeOH, catalyst and azeotroping agent are added and water is again azeotropically distilled off. (I) is useful as an intermediate, e.g. for highly active insecticides of the allethrin type. Carrying out the removal of water in two steps markedly increases the yield of (I).

Bactericidal concentrations of methylglyoxal produced by heated cells of Streptococcus faecalis

Abstract: Cells of Streptococcus faecalis that survived heating for 21 min at 60 °C were killed when resuspended at an initial cell density of about 1 × 108 viable units/mL and incubated at 33 °C for 24 h in a no-growth medium containing potassium phosphate buffer, pH 7.1, glucose, and casein hydrolysate. When such heated cells were resuspended at an initial cell density of about 1 × 107 viable units/mL or lower, subsequent cell death was reduced at least 10 000-fold. Unheated cells incubated under similar conditions at about 1 × 108 and 1 × 109 viable units/mL did not die. Cell death was due to a toxic compound synthesized by the heated cells, and supernatants from incubations showing a bactericidal effect contained a component, absent in nonlethal supernatants, that reacted with 2,4-dinitrophenylhydrazine. Thin-layer chromatography, mass spectrometer analysis, and the visible spectrum of the 2,4-dinitrophenylhydrazine derivative of the unknown and authentic methylglyoxal, and the positive response shown by the fr...