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(1/364) Different foci for the regulation of the activity of the KefB and KefC glutathione-gated K+ efflux systems.

KefB and KefC are glutathione-gated K+ efflux systems in Escherichia coli, and the proteins exhibit strong similarity at the level of both primary sequence and domain organization. The proteins are maintained closed by glutathione and are activated by binding of adducts formed between glutathione and electrophiles. By construction of equivalent mutations in each protein, this study has analyzed the control over inactive state of the proteins. A UV-induced mutation in KefB, L75S, causes rapid spontaneous K+ efflux but has only a minor effect on K+ efflux via KefC. Similarly amino acid substitutions that cause increased spontaneous activity in KefC have only small effects in KefB. Exchange of an eight amino acid region from KefC (HALESDIE) with the equivalent sequence from KefB (HELETAID) has identified a role for a group of acidic residues in controlling KefC activity. The mutations HELETAID and L74S in KefC act synergistically, and the activity of the resultant protein resembles that of KefB. We conclude that, despite the high degree of sequence similarity, KefB and KefC exhibit different sensitivities to the same site-specific mutations.  (+info)

(2/364) Methylglyoxal modification of protein. Chemical and immunochemical characterization of methylglyoxal-arginine adducts.

Methylglyoxal (MG), an endogenous metabolite that increases in diabetes and is a common intermediate in the Maillard reaction (glycation), reacts with proteins and forms advanced glycation end products. In the present study, we identify a novel MG-arginine adduct and also characterize the structure of a major fluorescent adduct. In addition, we describe the immunochemical study on the MG-arginine adducts using monoclonal antibody directed to MG-modified protein. Upon incubation of Nalpha-acetyl-L-arginine with MG at 37 degrees C, two nonfluorescent products and one fluorescent product were detected as the major products. The nonfluorescent products were identified as the Ndelta-(5-hydro-5-methyl-4-imidazolon-2-yl)-L-ornithine derivatives (5-hydro-5-methylimidazolone) and a novel MG-arginine adduct having a tetrahydropyrimidine moiety (Ndelta-(4-carboxy-4,6-dimethyl-5, 6-dihydroxy-1,4,5,6-tetrahydropyrimidine-2-yl)-L-ornithine). On the basis of the following chemical and spectroscopic evidence, the major fluorescent product, putatively identified as Ndelta-(5-methylimidazolon-2-yl)-L-ornithine (5-methylimidazolone), was found to be identical to Ndelta-(5-hydroxy-4, 6-dimethylpyrimidine-2-yl)-L-ornithine (argpyrimidine): (i) the low and high resolution fast atom bombardment-mass spectrometry gave a molecular ion peak at m/z of 297 (M+H) and a molecular formula of C10H25O6N4, respectively, which coincided with argpyrimidine; (ii) the 1H NMR spectrum of this product in d6-Me2SO showed a singlet at 2.10 ppm corresponding to six protons; (iii) the peak corresponding to the 5-methylimidazolone derivative was not detected by the liquid chromatography-mass spectrometry with the mode of selected ion monitoring; (iv) incubation of 5-hydro-5-methylimidazolone, a putative precursor of 5-methylimidazolone, at 37 degrees C for 14 days scarcely generated 5-methylimidazolone. On the other hand, as an immunochemical approach to the detection of these MG adducts, we raised the monoclonal antibodies (mAb3C and mAb6B) directed to the MG-modified protein and found that they specifically recognized the major fluorescent product, argpyrimidine, as the dominant epitope. The immunohistochemical analysis of the kidneys from diabetic patients revealed the localization of argpyrimidine in intima and media of small artery walls. Furthermore, the accumulation of argpyrimidine was also observed in some arterial walls of the rat brain after middle cerebral artery occlusion followed by reperfusion. These results suggest that argpyrimidine may contribute to the progression of not only long term diabetic complications, such as nephropathy and atherosclerosis, but also the tissue injury caused by ischemia/reperfusion.  (+info)

(3/364) Energetics of the proposed rate-determining step of the glyoxalase I reaction.

The proposed rate-limiting step of the reaction catalyzed by glyoxalase I is the proton abstraction from the C1 carbon atom of the substrate by a glutamate residue, resulting in a high-energy enolate intermediate. This proton transfer reaction was modelled using molecular dynamics and free energy perturbation simulations, with the empirical valence bond method describing the potential energy surface of the system. The calculated rate constant for the reaction is approximately 300-1500 s(-1) with Zn2+, Mg2+ or Ca2+ bound to the active site, which agrees well with observed kinetics of the enzyme. Furthermore, the results imply that the origin of the catalytic rate enhancement is mainly associated with enolate stabilization by the metal ion.  (+info)

(4/364) Simultaneous determination of formaldehyde and methylglyoxal in urine: involvement of semicarbazide-sensitive amine oxidase-mediated deamination in diabetic complications.

The deamination of methylamine and aminoacetone by semicarbazide-sensitive amine oxidase (SSAO) produces formaldehyde and methylglyoxal, respectively, which have been presumed to be involved in diabetic complications. A high-performance liquid chromatography procedure using 2,4-dinitrophenylhydrazine (DNPH) as a derivatizing agent is developed to determine endogenous formaldehyde, methylglyoxal, malondialdehyde, and acetaldehyde. The devised DNPH method is sensitive enough to analyze aldehyde levels in urine. An increase in the excretion of formaldehyde, methylglyoxal, and malondialdehyde is confirmed in streptozotocin-induced diabetic rats. Following the chronic administration of methylamine, the urinary levels of both formaldehyde and malondialdehyde (a product from lipid peroxidation) are found to be substantially increased. A potent selective SSAO inhibitor, (E)-2-(4-fluorophenethyl)-3-fluoroallylamine hydrochloride (MDL-72974A), reduced the formation of formaldehyde, methylglyoxal, and malondialdehyde. The increase of the cytotoxic aldehyde levels as a result of increased SSAO-mediated deamination may occur in some pathological conditions.  (+info)

(5/364) Induction of 1,2-dicarbonyl compounds, intermediates in the formation of advanced glycation end-products, during heat-sterilization of glucose-based peritoneal dialysis fluids.

OBJECTIVE: To study the presence of 1,2-dicarbonyl compounds in peritoneal dialysis (PD) fluids, their concentration in effluents with increasing dwell time, and their role in the formation of advanced glycation end-products (AGEs). MEASUREMENTS: Dicarbonyl compounds in heat- and filter-sterilized PD fluids were quantified by reverse-phase high performance liquid chromatography (HPLC) after derivatization to dimethoxyquinoxaline derivatives. Kinetics of the in vitro formation of AGEs upon incubation of 1,2-dicarbonyl compounds or PD fluids with albumin, with or without aminoguanidine, were measured by AGE fluorescence (excitation/emission wavelengths of 350 nm/430 nm). PATIENTS: AGEs and dicarbonyl compounds were measured in effluents collected from standardized 4-hour dwells from 8 continuous cycling peritoneal dialysis patients. RESULTS: In PD fluids, 3-deoxyglucosone (3-DG) has been identified as the major dicarbonyl compound formed during the process of heat sterilization. The process also formed glyoxal (GO) and methylglyoxal (MGO), with the amount of 3-DG being approximately 25-60 times higher than GO and MGO. When incubated with albumin, the identified 1,2-dicarbonyl compounds rapidly formed AGEs. The formation of AGEs was more pronounced in conventional heat-sterilized PD fluids compared with filter-sterilized PD fluids, and was completely inhibited by aminoguanidine. In effluents, the concentration of MGO, GO, and 3-DG decreased with increasing dwell time, with a concomitant increase in AGE fluorescence. CONCLUSIONS: The dicarbonyl compounds 3-DG, MGO, and GO are potent promoters of AGE formation. The presence of these and possibly other dicarbonyl compounds formed during heat sterilization of glucose-based PD fluids is, to a large extent, responsible for the in vitroAGE formation by these fluids, as evidenced by the speed of AGE formation in PD fluids and the complete inhibition by aminoguanidine. Because 3-DG, MGO, and GO are rapidly cleared from PD fluids during dialysis, these compounds may contribute to the in vivo AGE formation in PD patients.  (+info)

(6/364) Formation of glyoxal, methylglyoxal and 3-deoxyglucosone in the glycation of proteins by glucose.

The glycation of proteins by glucose has been linked to the development of diabetic complications and other diseases. Early glycation is thought to involve the reaction of glucose with N-terminal and lysyl side chain amino groups to form Schiff's base and fructosamine adducts. The formation of the alpha-oxoaldehydes, glyoxal, methylglyoxal and 3-deoxyglucosone, in early glycation was investigated. Glucose (50 mM) degraded slowly at pH 7.4 and 37 degrees C to form glyoxal, methylglyoxal and 3-deoxyglucosone throughout a 3-week incubation period. Addition of t-BOC-lysine and human serum albumin increased the rate of formation of alpha-oxoaldehydes - except glyoxal and methylglyoxal concentrations were low with albumin, as expected from the high reactivity of glyoxal and methylglyoxal with arginine residues. The degradation of fructosyl-lysine also formed glyoxal, methylglyoxal and 3-deoxyglucosone. alpha-Oxoaldehyde formation was dependent on the concentration of phosphate buffer and availability of trace metal ions. This suggests that alpha-oxoaldehydes were formed in early glycation from the degradation of glucose and Schiff's base adduct. Since alpha-oxoaldehydes are important precursors of advanced glycation adducts, these adducts may be formed from early and advanced glycation processes. Short periods of hyperglycaemia, as occur in impaired glucose tolerance, may be sufficient to increase the concentrations of alpha-oxoaldehydes in vivo.  (+info)

(7/364) Glucose degradation product methylglyoxal enhances the production of vascular endothelial growth factor in peritoneal cells: role in the functional and morphological alterations of peritoneal membranes in peritoneal dialysis.

Peritoneal membrane permeability deteriorates in peritoneal dialysis (PD) patients. We test whether glucose degradation products (GDPs) in PD fluids, glyoxal, methylglyoxal and 3-deoxyglucosone, stimulate the production of vascular endothelial growth factor (VEGF), a factor known to enhance vascular permeability and angiogenesis. VEGF increased in cultured rat mesothelial and human endothelial cells exposed to methylglyoxal, but not to glyoxal or 3-deoxyglucosone. VEGF also increased in peritoneal tissue of rats given intraperitoneally methylglyoxal. VEGF and carboxymethyllysine (CML) (formed from GDPs) co-localized immunohistochemically in mesothelial layer and vascular walls of the peritoneal membrane of patients given chronic PD. By contrast, in the peritoneum of non-uremic subjects, VEGF was identified only in vascular walls, in the absence of CML. VEGF production induced by GDPs may play a role in the progressive deterioration of the peritoneal membrane.  (+info)

(8/364) Glutathione is involved in environmental stress responses in Rhizobium tropici, including acid tolerance.

The isolation of rhizobial strains which exhibit an intrinsic tolerance to acidic conditions has been reported and has facilitated studies on the basic mechanisms underlying acid tolerance. Rhizobium tropici strain CIAT899 displays a high intrinsic tolerance to acidity and therefore was used in this work to study the molecular basis of bacterial responses to acid conditions and other environmental stresses. We generated a collection of R. tropici CIAT899 mutants affected in acid tolerance using Tn5-luxAB mutagenesis, and one mutant strain (CIAT899-13T2), which fails to grow under acid conditions, was characterized in detail. Strain CIAT899-13T2 was found to contain a single Tn5-luxAB insertion in a gene showing a high degree of similarity with the Escherichia coli gshB gene, encoding the enzyme glutathione synthetase. Intracellular potassium pools and intracellular pH levels were found to be lower in the mutant than in the parent. The glutathione-deficient mutant was shown to be sensitive to weak organic acids, osmotic and oxidative stresses, and the presence of methylglyoxal. Glutathione restores responses to these stresses almost to wild-type levels. Our data show that in R. tropici the production of glutathione is essential for growth in extreme environmental conditions. The mutant strain CIAT899-13T2 induced effective nodules; however, it was found to be outcompeted by the wild-type strain in coinoculation experiments.  (+info)