Showing papers on "Lactoylglutathione lyase 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.

Physiological and biochemical characterization of glyoxalase I, a general marker for cell proliferation, from a soybean cell suspension.

Abstract: Using a strictly auxin-dependent soybean (Glycine max (L.) Merr.) cell suspension, we studied the correlation of auxin-dependent cell proliferation and the activity of glyoxalase I (S-lactoylglutathione-lyase EC 4.4.1.5.), an enzyme generally associated with cell proliferation in animal, microbial and, as reported recently, also plant systems. We found the activity of glyoxalase I to be modulated during the proliferation cycle, with a maximal activity between day 2 and day 4 of culture growth. After starving the culture of auxins for three subsequent periods, both the enzyme activity and cell growth could be re-initiated with auxin. Enzyme activity reached its maximum 1 d before cell number was at a maximum. The enzyme was purified to homogeneity and characterized.

Purification and characterization of a glutathione peroxidase from the Aloe vera plant.

Abstract: Extracts from the parenchymous leaf-gel of the Aloe vera plant (Aloe barbadensis Miller) were shown to contain glutathione peroxidase (GSHPx) activity. The activity was purified to homogeneity by ion exchange and gel filtration (FPLC) chromatography in the presence of 0.5 mM glutathione. The native enzyme has an apparent molecular weight of 62 kD as determined by gel filtration. In the presence of sodium dodecylsulfate (SDS), the molecular weight was estimated to be about 16 kD as determined by polyacrylamide-gel electrophoresis (SDS-PAGE). The native enzyme is proposed to be constituted of four identical subunits; it also contains one atom of selenium per subunit, as found with most glutathione peroxidases from animal sources. The Km values were determined to be 3.2 mM for glutathione and 0.26 mM for the hydroperoxide substrate, cumene hydroperoxide. The enzyme is competitively inhibited by N, S, bis-FMOC glutathione (Ki = 0.32 mM), a potent inhibitor of glyoxalase II. Inhibitors of glyoxalase I (e.g. S-octylglutathione) have no effect on the peroxidase activity.

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.

Catalytic and molecular properties of glyoxalase I.

Abstract: Biological function of the glyoxalase system The biological role of the glyoxalase system remains to be defined exactly [ 1, 21. It is well established that glyoxalase I is capable of forming S ( 2 hydroxyacy1)glutathione from most 2-oxoaldehydes in the presence of glutathione [Z]. The majority of the glutathione thiolesters formed in reactions catalysed by glyoxalase I are substrates for glyoxalase I1 and are hydrolysed by the latter enzyme to the free 2-hydroxyacids and glutathione. By the coupled reactions catalysed by glyoxalases I and 11, electrophilic and cytotoxic 2-oxoaldehydes are converted to less reactive chemical species. Several known enzymic reactions involving endobiotics as well as xenobiotics lead to the formation of 2-oxoaldehydes. It has been suggested therefore that the glyoxalase system should be considered as part of the ensemble of glutathione-dependent detoxication enzymes [2]. From an evolutionary standpoint, the glyoxalase system may have arisen as a complement to other enzymes involved in detoxifying the products of oxidative metabolism [3, 41. For the inactivation of the electrophilic 2-oxoaldehyde function glyoxalase I is the most important enzyme, since it catalyses the elimination of the reactive chemical group and the intermediate glutathione thiolester may be regarded as essentially non-toxic. Glyoxalase I has been isolated from unicellular as well as from multicellular organisms and in human tissues the enzyme has a widespread distribution [5, 61. It has been suggested also that the glutathione thiolester S-n-lactoylglutathione, produced by the glyoxalase I reaction, may have specific cellular functions in cell proliferation, differentiation and other processes [ l , 71. In this context it is interest-

Glyoxalase activities in human tumour cell lines in vitro.

Abstract: The activities of glyoxalase I (EC 4.4.1.5) and glyoxalase II (EC 3.1.2.6) were measured in 3 human non-malignant and 28 tumour cell lines. The activity of glyoxalase I was in the range 321-8751 mUnits/mg of protein. The glyoxalase I/glyoxalase II activity ratio was in the range 7-147; glyoxalase I activity was always markedly greater than glyoxalase activity. There was no significant difference between glyoxalase activities in non-malignant and tumour cell lines. The activity of glyoxalase I was within and above the range found in normal human tissues and the activity of glyoxalase II was within the range and below that found in normal human tissues.

Inhibitors of glyoxalase I: design, synthesis, inhibitory characteristics and biological evaluation.

Abstract: Glyoxalase I (EC 4.4.1 .S lactoylglutathione lyase) catalyses the formation of S-u-lactoylglutathione from methylglyoxal and glutathione [ 11. The molecular species that binds to the active site of glyoxalase I is the hemithioacetal, formed nonenzymically from methylglyoxal and reduced glutat hione, y-L-glutamyl-S-( 1 -hydroxy-2-oxopropyl)L-cysteinylglycine. The isomerization of the hemithioacetal to S-1 Aactoylglutathione in the active site of glyoxalase I is thought to proceed via an endiolate intermediate. The reaction mechanism involves a shielded proton transfer from C-1 to C-2 of the hemithioacetal and rapid ketonization to the thioester product [2]. The S-u-lactoylglutathione formed is optically pure; generally, S(R)-2hydroxyacylglutathione derivatives are formed from glutathione and a-oxoaldehydes [3]. Recent evidence suggests that both Rand S-stereoisomers of the hemithioacetal bind to the active site of glyoxalase I and are converted to enediolate intermediates followed by stereospecific protonation to form the thioester [4]. The active site of glyoxalase I contains a glutathione-recognition site, a general-base catalyst (thought to be the imidazole moiety of a histidine residue [S]) and a proximate zinc ion, Zn2+, with co-ordinated water molecules that interact with the hemithioacetal and enediolate intermediate. The glutathione-recognition site is thought to form a complementary surface of counter-charged groups, lysine (mammalian enzyme) or arginine (yeast enzyme) residues and aspartate or glutamate residues, which bind the glutathionyl moiety of the hemithioacetal substrate by salt-bridge linkages (Figure 1). The charge characteristics of the glutathionyl moiety and the structural characteristics of the putative enediolate mechanistic intermediate have been conserved in developing potent, competitive, substrate-analogue inhibitors and transitionstate analogue inhibitors.

Effect of Radiation on Glyoxalase I and Glyoxalase II Activities in Spleen and Liver of Mice

Abstract: Swiss albino mice (7-8 weeks old) were irradiated with different doses (0-25 Gy) of gamma-radiation at a dose-rate of 0.05 Gy/s. The specific activities of glyoxalase I (GI) and glyoxalase II (GII) were determined in the spleen and liver immediately and on the 3rd and 6th day postirradiation. The results indicate that the glyoxalase system is radiosensitive, particularly glyoxalase I whose activity was enhanced even at low doses (0.5 Gy). The magnitude and mode of the radiation effect depends on dose and tissue. The patterns of the GI/GII ratio in the liver and spleen was very similar when measured immediately after irradiation. The radiation effect on the glyoxalase system persists even in the postirradiation period and was inversely related to the dose-rate. GSH and caffeine increased and chlorpromazine decreased the radiation-induced activity of GI, but all three modifiers enhanced radiation-induced inactivation of GII. Since the glyoxalase system may play an important role in the regulation of cell division and differentiation, radiation effects on this system may have some biochemical consequences.

Inhibition of hepatic xenobiotic metabolism and of glutathione-dependent enzyme activities by zinc ethylene-bis-dithiocarbamate in the rabbit.

Abstract: Effects of either a single (300 mg/kg) or a subchronic (0.3 and 0.6% for 70 days) oral administration of a dithiocarbamate fungicide (zinc ethylene-bis-dithiocarbamate, zineb) on hepatic drug metabolism and on the activity of several glutathione-dependent enzymes were investigated in male New Zealand White rabbits. While a pronounced reduction in the rate of oxidative biotransformations occurred after either single or repeated exposure, both cytochrome P450 and total haem content were lowered following acute challenge to zineb. None of the experimental protocols affected microsomal carboxylesterase but induced a marked increase in glutathione content and none of the examined glutathione-dependent enzymes was altered by the single administration of zineb, whereas the subchronically exposed rabbits showed a fall in the activities of both total glutathione S-transferase and selenium-independent glutathione peroxidase. In the 0.6% treated animals, a decrease in class mu glutathione S-transferase and glyoxalase I, and an increase in thiol-transferase activities were also recorded. It is concluded that (1) zineb is able to selectively impair oxidative drug metabolism with possible different mechanism(s) according to the duration of the exposure, (2) only the subchronic treatment affects glutathione-dependent enzymes, (3) the decrease in glutathione S-transferase activity would seem to be ascribed to a direct interaction with the fungicide.

Chicken eggshell porphyrins and the glyoxalase pathway: Its possible physiological role

Abstract: 1. 1. The plasma 4,5-dioxovaleric acid (DOVA) levels of two different breeds of chicken were determined and found to be higher in the group with a higher porphyrin eggshell content. 2. 2. The erythrocyte and uterus glyoxalase II activity, investigated by means of a new spectrophotometric method, was found to be significantly higher in the group with a low porphyrin eggshell content. 3. 3. A comparative genetic study of two chicken populations, one with white and one with dark eggshells, showed different gene frequencies for the glyoxalase I polymorphism. 4. 4. An interpretation of these data suggests that the glyoxalase pathway may be involved in the metabolism of early porphyrin precursors.

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

The influence of porphyrogenic drugs on the glyoxalase enzymes.

Abstract: A variety of drugs, known to induce acute attacks in porphyric patients has been found to inhibit the glyoxalase pathway. Glyoxalase I is competitively inhibited by sulphadimidine, oxytetracycline, chloramphenicol, etc. Allylisopropyl acetamide (AIA) seems to inhibit glyoxalase II. This inhibition could play a contributing role in the overproduction of porphyrins in porphyria and thus help explain the mechanism of induction of porphyric attacks. The results indicate, that the Heme pathway and the glyoxalase cycle are closely connected.

Putative role of antioxidant enzymes and glyoxalases in carcinogenesis

Abstract: The effects various drugs exert on antioxidant enzyme and glyoxalase activity in rat livers were studied. All drugs tested provoked a marked reduction in glutathione peroxidase and a small drop in both glyoxalase I and II activity. It is hypothesized that the substances tested support tumour development by neutralizing organic peroxides, thereby favouring the oxidation of carcinogens and, as a consequence, the formation of metabolites that trigger neoplastic transformation. The reduction in glyoxalase activity is probably attributable to the enhanced cell proliferation induced by the treatment.