Showing papers on "Methylglyoxal published in 1979"

Suppression of the formation of polyamines and macromolecules by dl-α-difluoromethylornithine and methylglyoxal bis(guanylhydrazone) in phytohaemagglutinin-activated human lymphocytes

Abstract: 1. The activation of human peripheral blood lymphocytes by phytohaemagglutinin in vitro was accompanied by striking increases in the concentrations of the natural polyamines putrescine, spermidine and spermine. 2. The enhanced accumulation of polyamines could be almost totally abolished by dl-alpha-difluoromethylornithine, a newly discovered irreversible inhibitor of l-ornithine decarboxylase (EC 4.1.1.17), or by methylglyoxal bis(guanylhydrazone) {1,1'-[(methylethanediylidene)dinitrilo]diguanidine}, an inhibitor of S-adenosyl-l-methionine decarboxylase (EC 4.1.1.50). The inhibition of polyamine accumulation was associated with a marked suppression of DNA synthesis, which was partially or totally reversed by low concentrations of exogenous putrescine, spermidine, spermine and cadaverine and by higher concentrations of 1,3-diaminopropane. 3. In contrast with some earlier studies, we found that methylglyoxal bis(guanylhydrazone), at concentrations that were sufficient to prevent polyamine accumulation, also caused a clear inhibition of protein synthesis in the activated lymphocytes. Similar results were obtained with difluoromethylornithine. The decrease in protein synthesis caused by both compounds preceded the impairment of DNA synthesis. The inhibition of protein synthesis by difluoromethylornithine was fully reversed by exogenous putrescine, spermidine and spermine, and that caused by methylglyoxal bis(guanylhydrazone) by spermidine and spermine. In further support of the idea that the inhibition of protein synthesis by these compounds was related to the polyamine depletion, we found that difluoromethylornithine caused a dose-dependent decrease in the incorporation of [(14)C]leucine into lymphocyte proteins which closely correlated with the decreased concentrations of cellular spermidine. 4. Difluoromethylornithine and methylglyoxal bis(guanylhydrazone) also elicited a variable depression in the incorporation of [(3)H]uridine and [(14)C]adenine into total RNA. The apparent turnover of lymphocyte RNA remained essentially unchanged in spite of severe polyamine depletion brought about by difluoromethylornithine. 5. The present results, as well as confirming the anti-proliferative action of the inhibitors of polyamine biosynthesis, suggest that polyamine depletion may interfere with reactions at different levels of gene expression.

Comparison of glyoxalase I purified from yeast (Saccharomyces cerevisiae) with the enzyme from mammalian sources.

Abstract: Glyoxalase I from yeast (Saccharomyces cerevisiae) purified by affinity chromatography on S-hexylglutathione–Sepharose 6B was characterized and compared with the enzyme from rat liver, pig erythrocytes and human erythrocytes. The molecular weight of glyoxalase I from yeast was, like the enzyme from Rhodospirillum rubrum and Escherichia coli, significantly less (approx. 32000) than that of the enzyme from mammals (approx. 46000). The yeast enzyme is a monomer, whereas the mammalian enzymes are composed of two very similar or identical subunits. The enzymes contain 1Zn atom per subunit. The isoelectric points (at 4°C) for the yeast and mammalian enzymes are at pH7.0 and 4.8 respectively; tryptic-peptide `maps' display corresponding dissimilarities in structure. These and some additional data indicate that the microbial and the mammalian enzymes may have separate evolutionary origins. The similarities demonstrated in mechanistic and kinetic properties, on the other hand, indicate convergent evolution. The kcat. and Km values for the yeast enzyme were both higher than those for the enzyme from the mammalian sources with the hemimercaptal adduct of methylglyoxal or phenylglyoxal as the varied substrate and free glutathione at a constant and physiological concentration (2mm). Glyoxalase I from all sources investigated had a kcat./Km value near 107s−1·m−1, which is close to the theoretical diffusion-controlled rate of enzyme–substrate association. The initial-velocity data show non-Michaelian rate saturation and apparent non-linear inhibition by free glutathione for both yeast and mammalian enzyme. This rate behaviour may have physiological importance, since it counteracts the effects of fluctuations in total glutathione concentrations on the glyoxalase I-dependent metabolism of 2-oxoaldehydes.

Relationship between inhibition of polyamine biosynthesis and DNA replication in activated lymphocytes

Abstract: This paper examines the relationship between inhibition of DNA replication and reduced polyamine levels in bovine lymphocytes using several different inhibitors of polyamine biosynthesis and various administration regimens. Dose-response curves for inhibition of DNA synthesis by methylglyoxal bis(guanylhydrazone) failed to provide evidence for an intracellular inhibitory site of this drug aside from polyamine biosynthesis. Experiments with two analogs of methylglyoxal bis(guanylhydrazone) showed a correlation between inhibition of DNA replication and lowered levels of spermidine and spermine. A new inhibitor of polyamine biosynthesis, α-difluoromethylornithine, which irreversibly inhibits ornithine decarboxylase and is structurally unrelated to methylglyoxal bis(guanylhydrazone), specifically inhibited DNA replication without altering the number of cells in S phase or the rates of protein and RNA synthesis. This physiological response was identical to that observed previously when polyamine synthesis was blocked with methylglyoxal bis(guanylhydrazone). We conclude that the observed inhibition of [ 3 H]thymidine incorporation was due to depressed intracellular polyamine levels and not to other pharmacological effects of the inhibitors that were unrelated to inhibition of polyamine biosynthesis. In the polyamine-deficient cells, inhibition was also observed when [ 3 H]thymidine incorporation was measured under conditions where de novo deoxythymidine 5′-phosphate synthesis was inhibited by amethopterin. It therefore appears that the observed inhibition was due to a depressed rate of DNA replication rather than to altered specific radioactivity of the intracellular DNA precursor, deoxythymidine 5′-triphosphate. Under the polyamine-deficient conditions, a lowered intracellular pool of deoxythymidine 5′-triphosphate was observed. However, it is concluded that this could not bring about the defects in replication observed and that polyamine deficiency probably induces defects in the nuclear replication machinery per se .

Effects of S -lactoylglutathione and inhibitors of glyoxalase I on histamine release from human leukocytes

Abstract: THE enzyme responsible for the conversion of methylglyoxal to lactic acid by animal tissues was first named glyoxalase1,2. It was later shown that there are two glyoxalase enzymes3. The first (glyoxalase I). converts methylglyoxal and reduced glutathione to S-lactoylglutathione and the second (glyoxalase II). converts this compound to D-lactic acid, regenerating glutathione in the process. These enzymes are very widely distributed4 and as yet have no clearly defined function(s). I describe here the enhancement of anti-IgE-induced histamine release by S-lactoylglutathione and its inhibition by an inhibitor of and an alternative substrate for glyoxalase I. These two compounds should, of course, decrease the production of endogenous S-lactoylglutathione. These experiments were undertaken for two reasons. First, a study from my laboratory has provided evidence that S-lactoylglutathione modulates microtubule assembly in vitro5 while the release of histamine from leukocytes is a secretory process thought to require an intact microtubule system6,7. Second, concanavalin A has been shown to cause histamine release from human leukocytes8,9 and has also been found to activate the two glyoxalase enzymes in lymphocytes and polymorphonuclear leukocytes10.

Polyamine replacement by magnesium ions in BHK-21/C13 cells.

Abstract: Cultures of BHK-21/C13 cells, whose growth was inhibited by deprivation of serum, were stimulated to grow by addition of serum to the culture medium. Addition of MgCl(2) to the medium, to increase the concentration of Mg(2+) ions by 15mm, 30min before addition of serum, had no effect on the stimulation of cell growth, but inhibited the accumulation of cellular spermidine, so that the spermidine/spermine molar ratio was lower in these cultures than in cultures that had received no additional cations. The increase in the activity of ornithine decarboxylase that occurs 4-5h after serum ;step-up' was substantially diminished by increasing the concentration of Mg(2+) ions, but not of Na(+) or K(+) ions, in the medium by 30mm, 30min before addition of serum, and this inhibition was maintained for at least 24h. Methylglyoxal bis(guanylhydrazone), added to serum-deprived cultures to a concentration of 20mum, 30min before addition of serum, severely inhibited the increase in cell growth. The inhibitory effects of the drug were prevented by simultaneous addition of spermidine to the medium (to 100mum), and were partly prevented by the simultaneous addition of Mg(2+) ions (to 30mm). Mg(2+) ions were particularly effective in overcoming the inhibitory effect of methylglyoxal bis(guanylhydrazone) on the synthesis of DNA. Thus although a certain lack of specificity for cations exists in BHK-21/C13 cells, in that Mg(2+) ions can be substituted for polyamines, particularly spermidine, to some extent, there are cellular processes for which the requirement for polyamines as cations is specific.

Quantum chemical calculations of model systems for ascorbic acid adducts with Schiff bases of lysine side chains: possibility of internal charge transfer in proteins

Abstract: Ab initio self-consistent field calculations for neutral and cationic ascorbic acid and model compounds have been performed. Furthermore, the bicyclic addition products of alpha-hydroxytetronic acid with methylglyoxal and with the Schiff base formed between methylglyoxal and methylamine have been calculated, again in their neutral and cationic forms, respectively. The results indicate that the investigated cations can act as strong electron acceptors. With the help of space-filling molecular models it has been demonstrated that such conformations of the Schiff base formed between the primary amino group of lysine side chains in proteins and the ascorbic acid methylglyoxal acetal are possible in which the lactone carbonyl group comes near to the N atoms of the peptide groups, so that charge can be transferred between these subunits.