Molecular characterization of glyoxalase-I from a higher plant; upregulation by stress

A cDNA, GLX1, encoding glyoxalase-I was isolated by differential screening of salt-induced genes in tomato. Glyoxalases-I and-II are ubiquitous enzymes whose functions are not clearly understood. They may serve to detoxify methylglyoxal produced from triosephosphates in all cells. The protein encoded by GLX1 shared 49.4% and 58.5% identity with glyoxalase-I isolated from bacteria and human, respectively. Furthermore, yeast cells expressing GLX1 showed a glyoxalase-I specific activity 20-fold higher than non-transformed cells. Both GLX1 mRNA and glyoxalase-I polypeptide levels increased 2- to 3-fold in roots, stems and leaves of plants treated with either NaCl, mannitol, or abscisic acid. Immunohistochemical localization indicated that glyoxalase-I was expressed in all cell types, with preferential accumulation in phloem sieve elements. This expression pattern was not appreciably altered by salt-stress. We suggest that the increased expression of glyoxalase-I may be linked to a higher demand for ATP generation and to enhanced glycolysis in salt-stressed plants.     

Protein kinase activities in Neurospora crassa

Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) has been purified from human erythrocytes using a simple chromatographic procedure. Purified enzyme was obtained from individuals who were homozygous for the principal isozyme (ADA 1) as well as from individuals who were heterogyzous for the major variant (ADA 2-1). Although ADA 1 and ADA 2-1 are electrophoretically distinguishable, they have many common physical and catalytic properties. No significant differences between the two isozymic forms were found in measurements of molecular weight, catalytic activity in the presence of various substrates and inhibitors, pH optimum, turnover number, and stability in conditions of both high and low pH. ADA 2-1 was, however, substantially less stable than ADA 1 with respect to thermal denaturation. These studies support the idea that adenosine deaminase activity in erythrocytes is lower in those individuals who possess the variant form of the enzyme.     

Characterization of two new electrophoretic variants of human triosephosphate isomerase: Stability,kinetic, and immunological properties

Two new electrophoretic variants of human triosephosphate isomerase (TPI) have been partially purified and characterized. The TPI Manchester variant, a cathodally migrating electrophoretic allozyme identified in an individual with the phenotype TPI 1-Manchester, is associated with a normal level of enzyme activity in erythrocytes and normal kinetic properties. It is very thermolabile at 55 and 57° C, although it is not uniquely sensitive to either guanidine-HCl or urea denaturation. The TPI Hiroshima-2 variant is an anodally migrating allozyme (the phenotype of proband is TPI 1-Hiroshima-2) with normal activity and kinetic properties and also normal stability characteristics. It is inactivated less by antisera raised against normal human TPI than either the normal or the Manchester allozyme. Dissociation-reassociation experiments utilizing these allozymes have confirmed that normal human red blood cell TPI isozymes are produced by a sequence of reactions (presumably deamidations) involving alternating subunits.Financial support was derived from Contract EY-77-C-02-2828 from the Department of Energy.     

Increased glyoxalase I levels inhibit accumulation of oxidative stress and an advanced glycation end product in mouse mesangial cells cultured in high glucose

Chronic high glucose levels lead to the formation of advanced glycation end-products (AGEs) as well as AGE precursors, such as methylglyoxal (MG) and glyoxal, via non-enzymatic glycation reactions in patients with diabetic mellitus. Glyoxalase 1 (GLO-1) detoxifies reactive dicarbonyls that form AGEs. To investigate the interaction between AGEs and GLO-1 in mesangial cells (MCs) under diabetic conditions, AGE levels and markers of oxidative stress were measured in GLO-1-overexpressing MCs (GLO-1-MCs) cultured in high glucose. Furthermore, we also examined levels of high glucose-induced apoptosis in GLO-1-MCs. In glomerular MCs, high glucose levels increased the formation of both MG and argpyrimidine (an MG-derived adduct) as well as GLO-1 expression. GLO-1-MCs had lower intracellular levels of MG accumulation, 8-hydroxy-deoxyguanosine (an oxidative DNA damage marker), 4-hydroxyl-2-nonenal (a lipid peroxidation product), and nitrosylated protein (a marker of oxidative-nitrosative stress) compared to control cells. Expression of mitochondrial oxidative phosphorylation complexes I, II, and III was also decreased in GLO-1-MCs. Furthermore, fewer GLO-1-MCs showed evidence of apoptosis as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick labeling assay, and activation of both poly (ADP-ribose) polymerase 1 cleavage and caspase-3 was lower in GLO-1-MCs than in control cells cultured in high glucose. These results suggest that GLO-1 plays a role in high glucose-mediated signaling by reducing MG accumulation and oxidative stress in diabetes mellitus.     

A novel mutation at the JK locus causing Jknull phenotype in a Chinese family

Jk (kidd) blood group antigens are carried by the urea transporter UT-B[1,2]. The Jknull phenotype, lack-ing urea permeability in erythrocytes[3,4], has a very low frequency in all populations except Polynesians and Finns[5]. In Japan, only 14 individuals with Jk (a-b-) phenotype were identified from 638460 screened donor’s blood samples using the 2 mol/L urea solution hemolysis test[6]. The frequency of Jknull is 0.27% in Polynesian, about 0.03% in Finland[7], and extremely rare in Fran…     

Generation of a GLO-2 deficient mouse reveals its effects on liver carbonyl and glutathione levels

ObjectiveHydroxyacylglutathione hydrolase (aka as GLO-2) is a component of the glyoxalase pathway involved in the detoxification of the reactive oxoaldehydes, glyoxal and methylglyoxal. These reactive metabolites have been linked to a variety of pathological conditions, including diabetes, cancer and heart disease and may be involved in the aging process. The objective of this study was to generate a mouse model deficient in GLO-2 to provide insight into the function of GLO-2 and to determine if it is potentially linked to endogenous oxalate synthesis which could influence urinary oxalate excretion.MethodsA GLO-2 knock out mouse was generated using CRISPR/Cas 9 techniques. Tissue and 24-h urine samples were collected under baseline conditions from adult male and female animals for biochemical analyses, including chromatographic measurement of glycolate, oxalate, glyoxal, methylglyoxal, D-lactate, ascorbic acid and glutathione levels.ResultsThe GLO-2 KO animals developed normally and there were no changes in 24-h urinary oxalate excretion, liver levels of methylglyoxal, glyoxal, ascorbic acid and glutathione, or plasma d-lactate levels. GLO-2 deficient males had lower plasma glycolate levels than wild type males while this relationship was not observed in females.ConclusionsThe lack of a unique phenotype in a GLO-2 KO mouse model under baseline conditions is consistent with recent evidence, suggesting a functional glyoxalase pathway is not required for optimal health. A lower plasma glycolate in male GLO-2 KO animals suggests glyoxal production may be a significant contributor to circulating glycolate levels, but not to endogenous oxalate synthesis.     

Half-site reactivity in 6-phosphogluconate dehydrogenase from human erythrocytes.

6-Phosphogluconate dehydrogenase from human erythrocytes was purified by an improved procedure. Binding studies showed that the dimeric enzyme binds 2 mol of NADP+/mol but only 1 mol of NADPH/mol, and that the bindings of oxidized and reduced coenzyme are mutually exclusive. From initial-rate kinetics and inhibition studies, a sequential random-order mechanism is proposed. Double-reciprocal plots with NADP+ as varied substrate show a downward curvature, indicating a negative co-operativity. We suggest that the negative co-operativity observed kinetically is a result of the half-site reactivity for the NADPH. The different binding stoichiometries for NADP+ and NADPH generate a non-linear relationship between the apparent dissociation constant for the NADPH and the concentrations of the NADP+, resulting in a regulatory mechanism highly sensitive to the changes in the NADP+/NADPH ratio.     

Caenorhabditis elegans HOPS and CCZ-1 mediate trafficking to lysosome-related organelles independently of RAB-7 and SAND-1

As early endosomes mature, the SAND-1/CCZ-1 complex acts as a guanine nucleotide exchange factor (GEF) for RAB-7 to promote the activity of its effector, HOPS, which facilitates late endosome–lysosome fusion and the consumption of AP-3–containing vesicles. We show that CCZ-1 and the HOPS complex are essential for the biogenesis of gut granules, cell type–specific, lysosome-related organelles (LROs) that coexist with conventional lysosomes in Caenorhabditis elegans intestinal cells. The HOPS subunit VPS-18 promotes the trafficking of gut granule proteins away from lysosomes and functions downstream of or in parallel to the AP-3 adaptor. CCZ-1 also acts independently of AP-3, and ccz-1 mutants mistraffic gut granule proteins. Our results indicate that SAND-1 does not participate in the formation of gut granules. In the absence of RAB-7 activity, gut granules are generated; however, their size and protein composition are subtly altered. These observations suggest that CCZ-1 acts in partnership with a protein other than SAND-1 as a GEF for an alternate Rab to promote gut granule biogenesis. Point mutations in GLO-1, a Rab32/38-related protein, predicted to increase spontaneous guanine nucleotide exchange, specifically suppress the loss of gut granules by ccz-1 and glo-3 mutants. GLO-3 is known to be required for gut granule formation and has homology to SAND-1/Mon1–related proteins, suggesting that CCZ-1 functions with GLO-3 upstream of the GLO-1 Rab, possibly as a GLO-1 GEF. These results support LRO formation occurring via processes similar to conventional lysosome biogenesis, albeit with key molecular differences.     

Assignment of a Mus musculus gene for triosephosphate isomerase to chromosome 6 and for glyoxalase-I to chromosome 17 using somatic cell hybrids

Chinese hamster X mouse hybrid cells segregating mouse chromosomes have been used to assign a gene for triosephosphate isomerase (TPI-1, EC 5.3.1.1, McKusick No. 19045) to mouse chromosome 6, and a gene for Glyoxalase-I (GLO-1, EC 4.4.1.5, McKusick No 13875) to mouse chromosome 17. The genes for TPI-1 and lactate dehydrogenase B are syntenic in man and probably so in the dog. It is therefore likely that they are syntenic also in the mouse. It is of interest then that there is a mouse gene, Ldr-1, on chromosome 6 that regulates the level of LDH B subunits in mouse erythrocytes. The locus for GLO-1 is closely linked to the major histocompatibility complex in man. Since the major histocompatibility complex in the mouse is present on chromosome 17, this locus and the Glo-1 locus are syntenic in the mouse as well. This finding adds to the number of autosomal gene pairs which are syntenic in both mouse and man and reinforces the belief that there is considerable conservation. of linkage groups during evolution.     

Genetic heterogeneity in adenosine deaminase (ADA) deficiency: five different mutations in five new patients with partial ADA deficiency.

Complete genetic deficiency of adenosine deaminase (ADA) results in a fatal syndrome of severe combined immunodeficiency (SCID). Genetic partial deficiency of ADA, with no detectable enzyme activity in erythrocytes but with variable amounts of enzyme activity detectable in other cells, is usually associated with normal immunologic function but can give rise to a late-onset, cellular immunodeficiency syndrome. We have previously described four different mutant alleles in four such partially ADA-deficient children. We have now examined ADA in lymphoid cells from five additional newly ascertained children with partial ADA deficiency with respect to electrophoretic mobility in starch gel, isoelectric point, heat-stability, and apparent Km and Vmax. These techniques identify at least five different abnormal alleles in these five additional unrelated subjects. Three of these abnormal alleles result in expression of abnormal allelic isozymes (allozymes) different from those previously described. These are: (1) an acidic allozyme that is less acidic than the acidic allozyme we have previously reported; (2) an allozyme that is even less acidic than (1); and (3) an allozyme with apparently normal charge but which is so heat sensitive that the lability to heat can easily be detected at physiologic to febrile temperatures. Two abnormal alleles detected in these five children could correspond with previously reported mutants. These are (4) a basic allozyme that could (but probably doesn't) correspond to the basic allozyme we have previously reported and (5) a "null" allele that cannot be differentiated by these methods from any other "null" allele seen in complete ADA- -SCIDs. Three of the five new patients are genetic compounds, identified either by the presence of two electrophoretically distinguishable allozymes or by family studies that demonstrate presence of a "null" allele in addition to an electrophoretically abnormal allozyme. In three patients, one or both allozymes are phenotypically indistinguishable from an abnormal allozyme also seen in a different individual. Determination of the nucleotide sequence will be required to determine whether or not the phenotypically indistinguishable mutations are indeed genotypically identical. The newly ascertained individuals appear to share a common ethnic West Indian background, out of proportion to the frequency of this ethnic background in the newborn population from which they were ascertained, suggesting that partial ADA deficiency may confer a selective advantage to the homozygous or heterozygous phenotype.     

Control of cell proliferation and differentiation by regulating polyamine biosynthesis in cultures of Brassica and its correlation with glyoxalase-I activity

Differentiation in Brussica cultures could be induced on basal medium lacking hormones, while addition of hormones (NAA, BA) resulted in profuse callusing without any differentiation. Supplementing the hormone medium with spermidine resulted in increase in the fresh weight and glyoxalase-I activity by 330% and 8-fold, respectively. Omission of hormones caused spermidine to be less effective in inducing either cell proliferation or differentiation. Methylglyoxal-bis(guanylhydrazone) (MGBG), an inhibitor of polyamine biosynthesis, had a retarding effect on callus induction and division of cells in suspension cultures but lead to differentiation and inhibited glyoxalase-I activity. The ability of spermidine to overcome MGBG enhanced differentiation was probably through the breaking of cell cycle arrest. Addition of glutathione, a coenzyme for glyoxalase-I enzyme, promoted cell division and enzyme activity both in callus and suspension cultures. pH emerged as an important factor in controlling glyoxalase-I activity and cell division. Results indicate involvement of spermidine in cell proliferation and differentiation and its correlation with glyoxalase-I activity.     

A Glyoxalase-1 Variant Associated with the t-Complex in House Mice

A quantitative variant of glyoxalase-1 associated with the t-complex in house mice is described. GLO-1C in red cell lysates from mice heterozygous for complementing t-haplotypes and from mice homozygous for the tw8-haplotype had less than one-third the GLO-1 activity of NZB/BlNJ, the inbred strain with the lowest activity previously reported. GLO-1C appeared to be determined by the structural locus Glo-1 and, together with two partial t6-haplotypes, was used to map Glo-1 to the telomeric portion of the t6-haplotype. Glo-1c was associated with all t-haplotypes tested and has not been found in mice that lack a t-complex. Thus, this variant of Glo-1c provides both a further example of gametic disequilibrium between the t-complex and linked loci and a readily identifiable marker for the t-complex.     

Biochemical characterization of aspartate aminotransferase allozymes from common wheat

Six allozymes of aspartate aminotransferase (AAT, EC 2.6.1.1): three plastidial (AAT-2 zone) and three cytosolic (AAT-3 zone) were isolated from common wheat (Triticum aestivum) seedlings and highly purified by a five-step purification procedure. The identity of the studied proteins was confirmed by mass spectrometry. The molecular weight of AAT allozymes determined by gel filtration was 72.4±3.6 kDa. The molecular weights of plastidial and cytosolic allozymes estimated by SDS-PAGE were 45.3 and 43.7 kDa, respectively. The apparent Michaelis constant (K _m) values determined for four substrates appeared to be very similar for each allozyme. The values of the turnover number (k _cat) and the k _cat/K _m ratio calculated for allozymes with L-aspartate as a leading substrate were in the range of 88.5–103.8 s^?1/10,412–10,795 s^?1 M^?1 for AAT-2 zone and 4.6–7.0 s^?1/527–700 s^?1 M^?1 for AAT-3 zone. These results clearly demonstrated much higher catalytic efficiency of AAT-2 allozymes. Therefore, partial sequences of cDNA encoding AATs from different zones were obtained using the RT-PCR technique. Comparison of the AAT-2 and AAT-3 amino acid sequences from active site regions revealed five non-conservative substitutions, which impact on the observed differences in the isozymes catalytic efficiency is discussed.     

Capillary electrophoresis of erythrocytes

Capillary electrophoresis (CE) of erythrocytes from different sources under various conditions is reported in this paper. It was found that erythrocyte samples from sheep, duck, and human showed characteristic and reproducible elution peaks, and that the retention times of A-, B-, AB-, and O-type erythrocytes from human blood were distinctively different; even subtle differences, among individuals with the same blood type could be detected by CE. A strictly linear correlation was obtained between the peak area and the amount of human erythrocyte over a range of 4.8 x 10(2)-1.9 x 10(4) cells (r=0.999), indicating that CE could be used for rapid and accurate quantification of erythrocytes. Using this CE protocol, the decrease of the surface electrical charge of erythrocyte during storage was confirmed. Therefore, this work demonstrated that CE could be a useful alternative for characterizing and quantifying erythrocytes or other cells.     

P blood group regulation of glycosphingolipid levels in human erythrocytes.

The neutral glycosphingolipid content of normal human erythrocytes was analyzed by a new method which utilizes high performance liquid chromatography. This rapid and accurate technique permits the quantitation of each of the major neutral glycolipids from individual blood samples. A correlation between the P blood group and the relative quantities of neutral glycosphingolipids is demonstrated. Erythrocytes from P1 individuals are shown to contain more globotriaosylceramide and less lactosylceramide than do erythrocytes from P2 individuals. The results of these experiments suggest the existence of a new phenotype in the P blood group system, and have further implications regarding the biosynthesis of the P blood group glycosphingolipids.     

Two pools of beta-endorphin-like immunoreactivity in blood: plasma and erythrocytes

While attempting to delineate the reason for the reported extreme variability of beta-endorphin-like immunoreactivity (beta-ir) in human plasma (eg., nondetectable to 1 ng/ml) by standard radioimmunoassay, we noted that a substantial portion of circulating beta-ir was associated with erythrocytes. That erythrocyte associated beta-ir is authentic beta-endorphin (beta-EP) was confirmed by high performance liquid chromatography (HPLC). Analysis of blood samples from rabbits, rats and mice revealed the presence of beta-ir in erythrocytes from these species as well. These results suggest that there are two pools of beta-endorphin-like immunoreactivity in blood: plasma and erythrocytes.     

Role of the Caenorhabditis elegans multidrug resistance gene, mrp-4, in gut granule differentiation

Caenorhabditis elegans gut granules are lysosome-related organelles with birefringent contents. mrp-4, which encodes an ATP-binding cassette (ABC) transporter homologous to mammalian multidrug resistance proteins, functions in the formation of gut granule birefringence. mrp-4(-) embryos show a delayed appearance of birefringent material in the gut granule but otherwise appear to form gut granules properly. mrp-4(+) activity is required for the extracellular mislocalization of birefringent material, body-length retraction, and NaCl sensitivity, phenotypes associated with defective gut granule biogenesis exhibited by embryos lacking the activity of GLO-1/Rab38, a putative GLO-1 guanine nucleotide exchange factor GLO-4, and the AP-3 complex. Multidrug resistance protein (MRP)-4 localizes to the gut granule membrane, consistent with it playing a direct role in the transport of molecules that compose and/or facilitate the formation of birefringent crystals within the gut granule. However, MRP-4 is also present in oocytes and early embryos, and our genetic analyses indicate that its site of action in the formation of birefringent material may not be limited to just the gut granule in embryos. In a search for genes that function similarly to mrp-4(+), we identified WHT-2, another ABC transporter that acts in parallel to MRP-4 for the formation of birefringent material in the gut granule.     

Flow cytometric analysis of erythrocyte populations in Tn syndrome blood using monoclonal antibodies to glycophorin A and the Tn antigen.

Flow cytometric analysis employing monoclonal antibodies to the Tn antigen and glycophorin A was used to characterize the erythrocyte populations present in blood samples from individuals with Tn syndrome. Four monoclonal antibodies specific for the Tn antigen, Gal-NAc monosaccharide, on human erythrocytes were obtained from a fusion of splenocytes from a Biozzi mouse immunized with red cells from a Tn individual. These monoclonal antibodies specifically recognize GalNAc monosaccharide sites located on the erythrocyte cell surface sialoglycoproteins, glycophorin A and glycophorin B, and do not bind to fixed normal red cells presenting the Neu-NAc alpha 2-3Gal beta 1-3(NeuNAc alpha 2-6)GalNAc alpha 1-O-Ser(Thr) tetrasaccharide or to fixed neuraminidase-digested cells presenting the Gal-GalNAc disaccharide. The percentages of Tn-positive red cells in samples from six unrelated Tn donors ranged from 28 to 99%. Binding of the glycophorin A-specific monoclonal antibodies showed that the erythrocytes composing the Tn-negative fraction presented normal amounts of the M and N epitopes on glycophorin A. The presumed somatic mutational origin of Tn-positive cells was tested in blood samples from five normal donors; three possible Tn cells were observed after analysis of a total of 1.1 x 10(7) erythrocytes, suggesting that the frequency of such cells in normal individuals is less than 1 x 10(-6).     

Growth rate correlates to individual heterozygosity in the European eel, Anguilla anguilla L

Heterozygosity-fitness correlations (HFCs) have been reported in populations of many species. We provide evidence for a positive correlation between genetic variability and growth rate at 12 allozyme loci in a catadromous marine fish species, the European eel (Anguilla anguilla L.). More heterozygous individuals show a significantly higher length and weight increase and an above average condition index in comparison with more homozygous individuals. To a lesser extent, six microsatellite loci show a similar pattern, with positive but not significant correlations between heterozygosity and growth rate. The HFCs observed could be explained by an effect of either direct allozyme over-dominance or associative overdominance. Selection affecting some of the allozyme loci would explain the greater strength of the HFCs found at allozymes in comparison with microsatellites and the lack of correlation between MLH at allozymes and MLH at microsatellites. Associative overdominance (where allozyme loci are merely acting as neutral markers of closely linked fitness loci) might provide an explanation for the HFCs if we consider that allozyme loci have a higher chance than microsatellites to be in linkage disequilibrium with fitness loci.     

Characterization of normal, glutathione-deficient and arginase-deficient sheep erythrocytes by 1H-NMR spectroscopy

Normal sheep erythrocytes as well as glutathione- (GSH-) deficient and arginase-deficient sheep erythrocytes have been characterized by 1H nuclear magnetic resonance spectroscopy. The GSH deficiency is a result of defective amino acid transport (lesion 1), diminished gamma-glutamylcysteine synthetase activity (lesion 2), or both (lesions (1 + 2)). 1H-NMR spectra of normal sheep erythrocytes are similar to those for human erythrocytes, and consist of resonances from a number of small intracellular molecules, including GSH. In contrast, the resonances for GSH in the GSH-deficient erythrocytes are much weaker, and strong resonances are observed for lysine, threonine and ornithine or arginine, depending on the arginase activity, in erythrocytes with lesion 1 and lesions (1 + 2). A comparison of the intensity of GSH resonances in spectra for normal and GSH-deficient erythrocytes with GSH levels determined spectrophotometrically following reaction with the nonspecific thiol reagent 5,5'-dithiobis(2-nitrobenzoate) (DTNB) indicates that either not all of the GSH determined with Ellman's reagent is free and observable by 1H-NMR or that not all of the thiol determined by Ellman's reagent is GSH. If the latter is the case, the GSH levels determined with Ellman's reagent for erythrocytes with lesions (1 + 2) are most affected, which might account for their high susceptibility to oxidative stress.