Purification and characterization of glyoxalase II from Hansenula mrakii

Glyoxalase II [S-(2-hydroxyacyl)glutathione hydrolase], one of the components of the glyoxalase system, catalyzes the hydrolysis of S-lactoylglutathione to glutathione and d-lactic acid. The enzyme was partially purified from the yeast Hansenula mrakii IFO 0895 by successive column chromatographies and polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 22,000 daltons by gel-filtration of Sephadex G-150 column chromatography and 24,000 daltons by SDS-polyacrylamide gel electrophoresis. The enzyme was specific to S-lactoyglutathione and S-acetylglutathione. The activity of the enzyme was strongly inhibited by Cu²⁺, p-chloromercuribenzoate and HgCl₂. The enzyme activity was also inhibited by hemimercaptal, a non-enzymatic condensation product between glutathione and methylglyoxal.     

Purification of formaldehyde and formate dehydrogenases from pea seeds by affinity chromatography and S-formylglutathione as the intermediate of formaldehyde metabolism

Formaldehyde dehydrogenase (EC 1.2.1.1) and formate dehydrogenase (EC 1.2.1.2) have been isolated in pure form from pea seeds by a rapid procedure which employs column chromatographies on 5′-AMP-Sepharose, Sephacryl S-200, and DE32 cellulose. The apparent molecular weights of formaldehyde and formate dehydrogenases are, respectively, 82,300 and 80,300 by gel chromatography, and they both consist of two similar subunits. The isoelectric point of formaldehyde dehydrogenase is 5.8 and that of formate dehydrogenase is 6.2. The purified formate dehydrogenase gave three corresponding protein and activity bands in electrophoresis and isoelectric focusing on polyacrylamide gel whereas formaldehyde dehydrogenase gave only one band. Formaldehyde dehydrogenase catalyzes the formation of S-formylglutathione from formaldehyde, and glutathione. Formate dehydrogenase can, besides formate, also use S-formylglutathione and two other formate esters as substrates. S-Formylglutathione has a lower K_m value (0.45 mm) than formate (2.1 mm) but the maximum velocity of S-formylglutathione is only 5.5% of that of formate. Pea extracts also contain a highly active S-formylglutathione hydrolase which has been separated from glyoxalase II (EC 3.1.2.6) and partially purified. S-Formylglutathione hydrolase is apparently needed between formaldehyde and formate dehydrogenases in the metabolism of formaldehyde in pea seeds, in contrast to what was recently reported for Hansenula polymorpha, a yeast grown on methanol.     

Glyoxalase II from Zea mays: properties and inhibition study of the enzyme purified by use of a new affinity ligand

The synthesis of N-(p-nitrocarbobenzoxy)glutathione (N-pNCBG) is reported. N-pNCBG and glutathione (GSH) were coupled to Affi-gel 10 by a thioester linkage and resulted in very effective bound ligands for a fast purification of glyoxalase II from corn. The S-(N-pNCBG)-affinity column showed a glyoxalase II binding capacity of up to 2-fold higher than that of the glutathione-affinity column. A single form of glyoxalase II was evidenced by PAGE in both crude extracts and in the affinity purified enzyme. A 45% recovery of glyoxalase II activity (purification, approx. 433-fold) was obtained for both matrices by a single chromatography. The purified glyoxalase is an acidic protein (pI 4.5) of about 26,000 relative molecular mass. Substrate studies for the corn glyoxalase II show, among possible substrates tested, that S-D-lactyl-glutathione is the preferred substrate. An inhibition study was performed with methyl-, propyl-, hexyl-, p-nitrobenzyl-, p-chlorophenacyl-, carbobenzoxy-, and p-nitrocarbobenzoxy-S-glutathione. Methyl-S-glutathione did not inhibit corn glyoxalase II; the others were found to be linear competitive inhibitors. The derivatives containing a thioether bond are weaker inhibitors than those containing a thioester bond or a carbonyl group. p-Nitrobenzyl-S-glutathione is the weakest inhibitor; the carbobenzoxy-S-derivatives are stronger inhibitors than the p-chlorophenacyl S-derivative.     

Purification by affinity chromatography of glutathione S-transferases A and C from rat liver cytosol

Purification of glutathione S-transferases A and C from rat liver cytosol using an affinity matrix coupled with cholic acid is described. The method provides a convenient means for the rapid and homogeneous preparation of both transferases.     

Photoaffinity labelling by S-(p-azidophenacyl)-glutathione of glyoxalase II and glutathione S-transferase

S-(p-azidophenacyl)-glutathione, $\text{l}$, is a linear competitive inhibitor at pH 7.40 of beef liver glyoxalase II with K_i = 7.96 × 10^?4 M. On irradiation at 340 nm it covalently inhibits glyoxalase II to a level of 42 ± 5% inhibition. This photoaffinity labelling is prevented by the presence of a glyoxalase II competitive inhibitor (the hemimercaptal of glutathione and methylglyoxal). A crude preparation of sheep liver glutathione S-transferases is also irreversibly inactivated (86% ± 5% inhibition) by irradiation at 320 nm in the presence of $\text{l}$.     

S-adenosylmethionine-dependent inactivation and radiolabeling of 1-aminocyclopropane-1-carboxylate synthase isolated from tomato fruits

1-Aminocyclopropane-1-carboxylic acid (ACC) synthase was partially purified from the homogenate of wounded tomato (Lycoperiscon esculentum Mill.) pericarp tissue by (NH₄)₂SO₄ fractionation followed by conventional column chromatography with diethylaminoethyl-Sepharose, Sephadex G-150, Affi-Gel blue and hydroxylapatite. The partially purified ACC synthase preparation attained a specific activity of about 12,000 nmoles per hour per milligram protein. Employing this enzyme preparation, we confirmed that the ACC synthase was inactivated by its substrate, S-adenosyl-l-methionine (SAM), during its catalytic action. When the partially purified enzyme preparation was incubated with [3,4-¹⁴C]SAM and the resulting proteins were analyzed by sodium dodecyl sulfate-gel electrophoresis, only one radioactive protein band was observed. This protein was thought to be ACC synthase based on its molecular mass of 50 kD and on the fact that it was specifically bound to a monoclonal antibody against ACC synthase (AB Bleecker et al. 1986 Proc Natl Acad Sci USA 83, 7755-7759). These results suggest that the substrate SAM acts as an enzyme-activated inactivator of ACC synthase by covalently linking a fragment of SAM molecule to the active site of ACC synthase, resulting in the inactivation of the enzyme.     

Paclobutrazol mitigates salt stress in <Emphasis Type="Italic">indica</Emphasis> rice seedlings by enhancing glutathione metabolism and glyoxalase system

Stress-induced methylglyoxal (MG) functions as a toxic molecule, inhibiting plant physiological processes such as photosynthesis and antioxidant defense systems. In the present study, an attempt was made to investigate the MG detoxification through glutathione metabolism in indica rice [Oryza sativa L. ssp. indica cv. Pathumthani 1] under salt stress by exogenous foliar application of paclobutrazol (PBZ). Fourteen-day-old rice seedlings were pretreated with 15 mg L^?1 PBZ foliar spray. After 7 days, rice seedlings were subsequently exposed to 0 (control) or 150 mM NaCl (salt stress) for 12 days. Prolonged salt stress enhanced the production of MG molecules and the oxidation of proteins, leading to decreased activity of glyoxalase enzymes, glyoxalase I (Gly I) and glyoxalase II (Gly II). Consequently, the decreased glyoxalase activities were also associated with a decline in reduced glutathione (GSH) content and glutathione reductase (GR) activity. PBZ pretreatment of rice seedlings under salt stress significantly lowered MG production and protein oxidation, and increased the activities of both Gly I and Gly II. PBZ also increased GSH content and GR activity along with the up-regulation of glyoxalase enzymes, under salt stress. In summary, salinity induced a high level of MG and the associated oxidative damage, while PBZ application reduced the MG toxicity by up-regulating glyoxalase and glutathione defense system in rice seedlings.     

Purification of S-2-hydroxyacylglutathione hydrolase (Glyoxalase II) from calf brain

S-2-Hydroxyacylglutathione hydrolase (Glyoxalase II) from calf brain has been purified 8333-times compared to 65,000 g supernatant of brain homogenate. The purification procedure employs Affi-Gel blue and preparative isoelectric focussing and offers a suitable method for the preparation of highly purified enzyme. Calf brain Glyoxalase II is a basic protein with a pl of 7.63 determined by isoelectric focusing. An evaluation of the relative molecular mass by gel filtration gave a value of about 23,000. During the purification procedure a constant Km value of about 0.325 mM was observed. A turnover number of 16,100 min-1 was calculated for the purified enzyme.     

Cysteine and glutathione in orange juice

The major part of the sulfhydryl compounds of both Valencia and Navel orange juice was found to exist as cysteine and glutathione. These two compounds were isolated and analyzed as their crystalline S-benzyl derivatives from concentrates prepared by preferential mercuric ion precipitation. Cysteine analysis before and after hydrolysis showed that all of the sulfhydryl of the concentrates was cysteine and glutathione. The glutathione analysis was confirmed by Woodward's glyoxalase method.     

Exogenous jasmonic acid modulates the physiology,antioxidant defense and glyoxalase systems in imparting drought stress tolerance in different Brassica species

This study examined the ability of jasmonic acid (JA) to enhance drought tolerance in different Brassica species in terms of physiological parameters, antioxidants defense, and glyoxalase system. Ten-day-old seedlings were exposed to drought (15 % polyethylene glycol, PEG-6000) either alone or in combination with 0.5 mM JA. Drought significantly increased lipoxygenase activity and oxidative stress, levels of malondialdehyde and H₂O₂. Drought reduced seedling biomass, chlorophyll (chl) content, and leaf relative water content (RWC). Drought increased proline, oxidized ascorbate (DHA) and glutathione disulfide (GSSG) levels. Drought affected different species differently: in B. napus, catalase (CAT) and glyoxalase II (Gly II) activities were decreased, while glutathione-S-transferase (GST) and glutathione peroxidase (GPX) activities were increased in drought-stressed compared to unstressed plants; in B. campestris, activities of glutathione reductase (GR), glyoxalase I (Gly I), GST, and GPX were increased, monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), CAT and other enzymes were decreased; in B. juncea, activities of ascorbate peroxidase, GR, GPX, Gly I were increased; Gly II activity was decreased and other enzymes did not change. Spraying drought-stressed seedlings with JA increased GR and Gly I activities in B. napus; increased MDHAR activity in B. campestris; and increased DHAR, GR, GPX, Gly I and Gly II activities in B. juncea. JA improved fresh weight, chl, RWC in all species, dry weight increased only in B. juncea. Brassica juncea had the lowest oxidative stress under drought, indicating its natural drought tolerance capacity. The JA improved drought tolerance of B. juncea to the highest level among studied species.     

Alleviation of osmotic stress in Brassica napus,B. campestris,and B. juncea by ascorbic acid application

The roles of ascorbic acid (AsA, 1 mM) under an osmotic stress [induced by 15 % (m/v) polyethylene glycol, PEG-6000] were investigated by examining morphological and physiological attributes in Brassica species. The osmotic stress reduced the fresh and dry masses, leaf relative water content (RWC), and chlorophyll (Chl) content, whereas increased the proline (Pro), malondialdehyde (MDA), and H₂O₂ content, and lipoxygenase (LOX) activity. The ascorbate content in B. napus, B. campestris, and B. juncea decreased, increased, and remained unaltered, respectively. The dehydroascorbate (DHA) content increased only in B. napus. The AsA/DHA ratio was reduced by the osmotic stress in all the species except B. juncea. The osmotic stress increased the glutathione (GSH) content only in B. juncea, but increased the glutathione disulfide (GSSG) content and decreased the GSH/GSSG ratio in all the species. The osmotic stress increased the activities of ascorbate peroxidase (APX) (except in B. napus), glutathione reductase (GR) (except in B. napus), glutathione S-transferase (GST) (except in B. juncea), and glutathione peroxidase (GPX), and decreased the activities of catalase (CAT) and monodehydroascorbate reductase (MDHAR) (only in B. campestris). The osmotic stress decreased the glyoxalase I (Gly I) and increased glyoxalase II (Gly II) activities. The application of AsA in combination with PEG improved the fresh mass, RWC, and Chl content, whereas decreased the Pro, MDA, and H₂O₂ content in comparison with PEG alone. The AsA addition improved AsA-GSH cycle components and improved the activities of all antioxidant and glyoxalase enzymes in most of the cases. So, exogenous AsA improved physiological adaptation and alleviated oxidative damage under the osmotic stress by improving the antioxidant and glyoxalase systems. According to measured parameters, B. juncea can be recognized as more drought tolerant than B. napus and B. campestris.     

A rapid method for the purification of S-adenosylmethionine: Protein-carboxyl O-methyltransferase by affinity chromatography

A simple method to purify S-adenosylmethionine: protein-carboxyl O-methyltransferase (protein methylase II, EC 2.1.1.24) from calf brain has been developed using affinity chromatography. The product of the reaction, S-adenosyl-l-homocysteine, which is a competitive inhibitor of the enzyme, was covalently linked to Sepharose beads. This gel proved to be an effective binder for protein methylase II at pH 6.2 and allowed for specific removal of the enzyme by the addition of the methyl donor substrate, S-adenosyl-l-methionine to the elution buffer. One step using this affinity chromatography column resulted in 377-fold purification of the enzyme and 71% recovery of the activity. Subsequent Sephadex G-100 chromatography enabled the enzyme to be purified 3000-fold from the calf brain whole homogenate. The purified enzyme showed a number of protein methylase II activity peaks following preparative gel electrophoresis with one major enzyme peak.     

Glutathione in plants: biosynthesis and physiological role in environmental stress tolerance

Glutathione (GSH; γ-glutamyl-cysteinyl-glycine) is a small intracellular thiol molecule which is considered as a strong non-enzymatic antioxidant. Glutathione regulates multiple metabolic functions; for example, it protects membranes by maintaining the reduced state of both α-tocopherol and zeaxanthin, it prevents the oxidative denaturation of proteins under stress conditions by protecting their thiol groups, and it serves as a substrate for both glutathione peroxidase and glutathione S-transferase. By acting as a precursor of phytochelatins, GSH helps in the chelating of toxic metals/metalloids which are then transported and sequestered in the vacuole. The glyoxalase pathway (consisting of glyoxalase I and glyoxalase II enzymes) for detoxification of methylglyoxal, a cytotoxic molecule, also requires GSH in the first reaction step. For these reasons, much attention has recently been directed to elucidation of the role of this molecule in conferring tolerance to abiotic stress. Recently, this molecule has drawn much attention because of its interaction with other signaling molecules and phytohormones. In this review, we have discussed the recent progress in GSH biosynthesis, metabolism and its role in abiotic stress tolerance.     

Production of S-lactoylglutathione by glycerol-adapted Saccharomyces cerevisiae and genetically engineered Escherichia coli cells

Summary The enzymatic production of S-lactoylglutathione was studied by applying glyoxalase I to glycerol-grown cells of Saccharomyces cerevisiae and Escherichia coli cells dosed with Pseudomonas putida glyoxalase I gene. The glyoxalase I in S. cerevisiae cells was markedly induced when the cells were grown on glycerol. The activity of the enzyme in glycerol-grown cells was more than 20-fold higher compared with that of the glucose-grown cells. By using extracts of glycerol-grown yeast cells, about 5 mmol/1 (2 g/l) of S-lactoylglutathione was produced from 10 mM methylglyoxal and 50 mM glutathione within 1 h. The extracts of E. coli cells carrying a hybrid plasmid pGI423, which contains P. putida glyoxalase I gene, showed approximately 170-fold higher glyoxalase I activity than that of E. coli cells without pGI423. The extracts were used for production of S-lactoylglutathione and, under optimal conditions, about 40 mmol/l (15 g/l) of S-lactoylglutathione was produced from 50 mM methylglyoxal and 100mM glutathione within 1 h.     

Isolation of glyoxalase II from two different compartments of rat liver mitochondria. Kinetic and immunochemical characterization of the enzymes

Two separate pools of glyoxalase II were demonstrated in rat liver mitochondria, one in the intermembrane space and the other in the matrix. The enzyme was purified from both sources by affinity chromatography on S-(carbobenzoxy)glutathione-Affi-Gel 40. From both crude and purified preparations polyacrylamide gel-electrophoresis resolved multiple forms of glyoxalase II, two from the intermembrane space and five from the matrix. Among the thioesters of glutathione tested as substrates, S-D-lactoylglutathione was hydrolyzed most efficiently by the enzymes from both sources. Significant differences were observed in the specificities between the intermembrane space and matrix enzymes with S-acetoacetylglutathione, S-acetylglutathione, S-propionylglutathione and S-succinylglutathione as substrates. Pure glyoxalase II from rat liver cytosol was chemically polymerized and used as antigen. Antibodies were raised in rabbits and the antiserum was used for comparison of the two purified mitochondrial enzymes with cytosolic glyoxalase II by immunoblotting. The enzyme purified from the intermembrane space cross-reacted with the antiserum, but the matrix glyoxalase II did not. The results give evidence for the presence in rat liver mitochondria of two species of glyoxalase II with differing characteristics. Only the enzyme from the intermembrane space appears to resemble the cytosolic glyoxalase II forms.     

Production of S‐lactoylglutathione by high activity whole cell biocatalysts prepared by permeabilization of recombinant Saccharomyces cerevisiae with alcohols

The permeabilization of yeast cells with methanol, ethanol, and isopropyl alcohol under various conditions was studied to develop the preparation method of high activity whole cell biocatalysts. Recombinant Saccharomyces cerevisiae, which intracellularly overexpresses glyoxalase I and catalyzes the conversion of methylglyoxal to S‐lactoylglutathione in the presence of glutathione, was used as the model system. The permeabilization treatments with alcohols significantly enhanced the activities of yeast cells. Especially, the initial S‐lactoylglutathione production rates of cells permeabilized with 40% ethanol and isopropyl alcohol solutions for 10 min at 4°C were high and were 364 and 582 times larger than those of untreated cells, respectively. These permeabilized yeast cells retained high activities during repeated batch reactions. Even in third batch reaction, they showed approximately 70–80% of the activity in the first batch. The plasma membrane of S. cerevisiae cells was damaged by the treatment with alcohol solutions in such a way that leakage of glyoxalase I from the cells is rather small and that both substrate and product show very high permeability. The initial S‐lactoylglutathione production rates of these permeabilized cells were 1.5–2.5 times larger than those of glyoxalase I in cell extracts prepared by ethyl acetate method from the same amount of cells. These results demonstrate that the recombinant S. cerevisiae cells permeabilized with alcohol solutions under the optimum condition are very effective whole cell biocatalysts. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 54–60, 1999.     

The isolation of a fetal rat liver glutathione S-tranferase isoenzyme with high glutathione peroxidase activity

A previously uncharacterized glutathione S-transferase isoenzyme which is absent from normal adult rat livers has been isolated fetal rat livers. The enzyme was purified using a combination of affinity chromatography, CM-cellulose column chromatography and chromatofocusing. It is composed of two non-identical subunits, namely, subunit Y_c (M_r 28 000) and a subunit (M_r 25 500) recently reported by us to be uniquely present in fetal rat livers and which we now refer to as subunit ‘Y_fetus’. The enzyme which we term glutathione S-transferase Y_cY_fetus has an isoelectric point of approx. 8.65 and has glutathione S-transferase activity towards a number of substrates. The most significant property of the fetal isozyme is its high glutathione peroxidase activity towards the model substrate cumene hydroperoxide. We suggest that this isozyme serves a specific function in protecting fetuses against the possible teratogenic effects of organic peroxides.     

The sterochemical configuration of the lactoyl group of S-lactoylglutathionine formed by the action of glyoxalase I from porcine erythrocytes and yeast

S-Lactoylglutatione formed by the reaction between methylglyoxal and glutathione, catalyzed by glyoxalase I, has been isolated by means of gel filtration. The product was analyzed for content of thiolester, thiol, and d- and l-lactate before and after hydrolysis of the thiolester linkage. From the results it is concluded that glyoxalase I from both porcine erythrocytes and yeast stereospecifically transfers hydrogen to form S-d-lactoylglutathione from methylglyoxal and glutathione.     

Characterization of Glyoxalase I (E. coli)—Inhibitor Interactions by Electrospray Time-of-Flight Mass Spectrometry and Enzyme Kinetic Analysis

Potential inhibitors of the enzyme glyoxalase I from Escherichia coli have been evaluated using a combination of electrospray mass spectrometry and conventional kinetic analysis. An 11-membered library of potential inhibitors included a glutathione analogue resembling the transition-state intermediate in the glyoxalase I catalysis, several alkyl-glutathione, and one flavonoid. The E. coli glyoxalase I quaternary structure was found to be predominantly dimeric, as is the homologous human glyoxalase I. Binding studies by electrospray revealed that inhibitors bind exclusively to the dimeric form of glyoxalase I. Two specific binding sites were observed per dimer. The transition-state analogue was found to have the highest binding affinity, followed by a newly identified inhibitor; S-{2-[3-hexyloxybenzoyl]-vinyl}glutathione. Kinetic analysis confirmed that the order of affinity established by mass spectrometry could be correlated to inhibitory effects on the enzymatic reaction. This study shows that selective inhibitors may exist for the E. coli homologue of the glyoxalase I enzyme.     

Characterization of an endo-alpha-N-acetyl galactosaminidase from Diplococcus pneumoniae.

Evidence is presented for the presence in filtrates of Diplococcus pneumoniae of an endo-glycosidase capable of acting on the O-glycosidic linkage between N-acetyl galactosamine and serine or threonine residues. The glycosidase was partially purified by chromatography on Affi-Gel 202. The resulting preparation acted on glycopeptides from mouse melanoma, fetuin and pig submaxillary mucin to release a disaccharide characterized as galactosyl-N-acetyl galactosamine. The enzyme had no action on phenyl α-N-acetyl-D-galactosaminide, asialo ovine submaxillary mucin or monosialoganglioside. A similar activity was detected in a commercial preparation of Clostridium perfringens neuraminidase.