Measurement of glyoxalase activities

Arai, Makoto; Nihonmatsu-Kikuchi, Naomi; Itokawa, Masanari; Rabbani, Naila; Thornalley, Paul J.

doi:10.1042/bst20140010

Cited by 89 publications

(55 citation statements)

References 20 publications

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“…The activity of GLO1 was measured by determining the isomerisation rate of hemithioacetal, formed non-enzymatically by pre-incubation of MG and reduced glutathione (GSH), to S-D-lactoylglutathione, followed spectrophotometrically at 240 nm; Δε 240 = 2.86 mM −1 cm −1 22. GLO1 activity is given in U per mg protein.…”

Section: Methodsmentioning

confidence: 99%

Glyoxalase 1-knockdown in human aortic endothelial cells – effect on the proteome and endothelial function estimates

Stratmann

Engelbrecht

Espelage

et al. 2016

Sci Rep

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Methylglyoxal (MG), an arginine-directed glycating agent, is implicated in diabetic late complications. MG is detoxified by glyoxalase 1 (GLO1) of the cytosolic glyoxalase system. The aim was to investigate the effects of MG accumulation by GLO1-knockdown under hyperglycaemic conditions in human aortic endothelial cells (HAECs) hypothesizing that the accumulation of MG accounts for the deleterious effects on vascular function. SiRNA-mediated knockdown of GLO1 was performed and MG concentrations were determined. The impact of MG on the cell proteome and targets of MG glycation was analysed, and confirmed by Western blotting. Markers of endothelial function and apoptosis were assessed. Collagen content was assayed in cell culture supernatant. GLO1-knockdown increased MG concentration in cells and culture medium. This was associated with a differential abundance of cytoskeleton stabilisation proteins, intermediate filaments and proteins involved in posttranslational modification of collagen. An increase in fibrillar collagens 1 and 5 was detected. The extracellular concentration of endothelin-1 was increased following GLO1-knockdown, whereas the phosphorylation and amount of eNOS was not influenced by GLO1-knockdown. The expression of ICAM-1, VCAM-1 and of MCP-1 was elevated and apoptosis was increased. MG accumulation by GLO1-knockdown provoked collagen expression, endothelial inflammation and dysfunction and apoptosis which might contribute to vascular damage.

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Section: Methodsmentioning

confidence: 99%

Glyoxalase 1-knockdown in human aortic endothelial cells – effect on the proteome and endothelial function estimates

Stratmann

Engelbrecht

Espelage

et al. 2016

Sci Rep

Self Cite

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“…Enzyme activities were assayed at 37 C. GLO1 activity in frontal cortex lysates was assayed spectrophotometrically 29 by monitoring the appearance of S-D-lactoylglutathione at 240 nm (" ¼ 2.86 mM/cm) in a Shimadzu Instruments model UV-1800 spectrophotometer. A lysate-free reaction mixture containing hemithioacetal substrate exhibited no intrinsic reaction.…”

Section: Cerebrocortical Enzyme Activities and Protein Contentmentioning

confidence: 99%

Featured Article: Pyruvate preserves antiglycation defenses in porcine brain after cardiac arrest

Scott

Nguyen

Cherry

et al. 2017

Exp Biol Med (Maywood)

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Recent studies have demonstrated a pivotal role of protein glycation in brain injury. Methylglyoxal, a by-product of glycolysis and a powerful glycating agent in brain, is detoxified by the glutathione-catalyzed glyoxalase (GLO) system, but the impact of cardiac arrest (CA) and cardiocerebral resuscitation (CCR) on the brain's antiglycation defenses is unknown. This study in a swine model of CA and CCR demonstrated for the first time that the intense cerebral ischemia-reperfusion imposed by CA-resuscitation disabled glyoxalase-1 and glutathione reductase (GR), the source of glutathione for methylglyoxal detoxification. Moreover, intravenous administration of pyruvate, a redox-active intermediary metabolite and antioxidant in brain, prevented inactivation of glyoxalase-1 and GR and blunted protein glycation in cerebral cortex. These findings in a large mammal are first evidence of GLO inactivation and the resultant cerebral protein glycation after CA-resuscitation, and identify novel actions of pyruvate to minimize protein glycation in postischemic brain. AbstractCardiac arrest (CA) and cardiocerebral resuscitation (CCR)-induced ischemia-reperfusion imposes oxidative and carbonyl stress that injures the brain. The ischemic shift to anaerobic glycolysis, combined with oxyradical inactivation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), provokes excessive formation of the powerful glycating agent, methylglyoxal. The glyoxalase (GLO) system, comprising the enzymes glyoxalase 1 (GLO1) and GLO2, utilizes reduced glutathione (GSH) supplied by glutathione reductase (GR) to detoxify methylglyoxal resulting in reduced protein glycation. Pyruvate, a natural antioxidant that augments GSH redox status, could sustain the GLO system in the face of ischemia-reperfusion. This study assessed the impact of CA-CCR on the cerebral GLO system and pyruvate's ability to preserve this neuroprotective system following CA. Domestic swine were subjected to 10 min CA, 4 min closed-chest CCR, defibrillation and 4 h recovery, or to a non-CA sham protocol. Sodium pyruvate or NaCl control was infused (0.1 mmol/kg/min, intravenous) throughout CCR and the first 60 min recovery. Protein glycation, GLO1 content, and activities of GLO1, GR, and GAPDH were analyzed in frontal cortex biopsied at 4 h recovery. CA-CCR produced marked protein glycation which was attenuated by pyruvate treatment. GLO1, GR, and GAPDH activities fell by 86, 55, and 30%, respectively, after CA-CCR with NaCl infusion. Pyruvate prevented inactivation of all three enzymes. CA-CCR sharply lowered GLO1 monomer content with commensurate formation of higher molecular weight immunoreactivity; pyruvate preserved GLO1 monomers. Thus, ischemia-reperfusion imposed by CA-CCR disabled the brain's antiglycation defenses. Pyruvate preserved these enzyme systems that protect the brain from glycation stress.

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“…The change in absorbation (ΔE) at 240 nm by 37°C was measured for 1 hr to allow the reaching of the maximum value. GLO1 enzyme activity (A) in whole cell lysates was calculated as following:

A = false(V_{total} \times normalΔ E false) / false(V_{sample} \times ε_{Hemithioacetal} \times d false)

and A (whole protein lysate) = A / m with ε = 2.86 mM −1 *cm −1 (Arai, Nihonmatsu‐Kikuchi, Itokawa, Rabbani, & Thornalley, ) .…”

Section: Methodsmentioning

confidence: 99%

Glyoxalase 1 expression is downregulated in preimplantation blastocysts of diabetic rabbits

Seeling

Haucke

Grybel

et al. 2019

Reprod Domestic Animals

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In a diabetic pregnancy, an altered maternal metabolism led to increased formation of reactive α‐dicarbonyls such as glyoxal (GO) and methylglyoxal (MGO) in the reproductive organs and embryos. The enzyme glyoxalase (GLO) 1 detoxifies reactive α‐dicarbonyls thus protecting cells against malfunction or modifications of proteins by advanced glycated end products (AGEs). The aim of this study was to analyse the influence of a maternal insulin‐dependent diabetes mellitus (IDD) on GLO1 expression and activity in preimplantation embryos in vivo and human trophoblast cells (Ac‐1M88) in vitro. Maternal diabetes was induced in female rabbits by alloxan before conception and maintained during the preimplantation period. GLO1 expression and activity were investigated in 6‐day‐old blastocysts from healthy and diabetic rabbits. Furthermore, blastocysts and human trophoblast cells were exposed in vitro to hyperglycaemia, GO and MGO and analysed for GLO1 expression and activity. During gastrulation, GLO1 was expressed in all compartments of the rabbit blastocyst. Maternal diabetes decreased embryonic GLO1 protein amount by approx. 30 per cent whereas the enzymatic activity remained unchanged, indicating that the specific GLO1 activity increases along with metabolic changes. In in vitro cultured embryos, neither hyperglycaemia nor MGO and GO had an effect on GLO1 protein amount. In human trophoblast cells, a stimulating effect on the GLO1 expression was shown in the highest GO concentration, only. Our data show that maternal diabetes mellitus affects the specific activity of GLO1, indicating that GLO1 was post‐translationally modified due to changes in metabolic processes in the preimplantation embryos.

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Measurement of glyoxalase activities

Cited by 89 publications

References 20 publications

Glyoxalase 1-knockdown in human aortic endothelial cells – effect on the proteome and endothelial function estimates

Glyoxalase 1-knockdown in human aortic endothelial cells – effect on the proteome and endothelial function estimates

Featured Article: Pyruvate preserves antiglycation defenses in porcine brain after cardiac arrest

Glyoxalase 1 expression is downregulated in preimplantation blastocysts of diabetic rabbits

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