The Experts below are selected from a list of 222 Experts worldwide ranked by ideXlab platform

Barry J. Shelp - One of the best experts on this subject based on the ideXlab platform.

  • REVIEW ARTICLE Role of plant Glyoxylate Reductases during stress: a hypothesis
    2016
    Co-Authors: Wendy L. Allan, Shawn M. Clark, Gordon J. Hoover, Barry J. Shelp
    Abstract:

    Molecular modelling suggests that a group of proteins in plants known as the β-hydroxyacid dehydrogenases, or the hydroxyisobutyrate dehydrogenase superfamily, includes enzymes that reduce succinic semialdehyde and Glyoxylate to γ-hydroxybutyrate and glycolate respectively. Recent biochemical and expression studies reveal that NADPH-dependent cytosolic (termed GLYR1) and plastidial (termed GLYR2) isoforms of suc-cinic semialdehyde/Glyoxylate Reductase exist in Arabidopsis. Succinic semialdehyde and Glyoxylate are typically generated in leaves via two distinct metabolic pathways, γ-aminobutyrate and glycolate respectively. In the present review, it is proposed that the GLYRs function in the detoxification of both aldehydes during stress and contribute to redox balance. Outstanding questions are highlighted in a scheme for the subcellular organization of the detoxification mechanism in Arabidopsis. Key words: aldehyde detoxification, Glyoxylate Reductase, β-hydroxyacid dehydrogenase, redox balance, succinic semi

  • Glyoxylate Reductase isoform 1 is localized in the cytosol and not peroxisomes in plant cells.
    Journal of integrative plant biology, 2012
    Co-Authors: Steven L. K. Ching, Barry J. Shelp, Amanda Rochon, Satinder K. Gidda, Owen R. Van Cauwenberghe, Robert T. Mullen
    Abstract:

    Glyoxylate Reductase (GLYR) is a key enzyme in plant metabolism which catalyzes the detoxification of both photorespiratory Glyoxylate and succinic semialdehdye, an intermediate of the γ-aminobutyrate (GABA) pathway. Two isoforms of GLYR exist in plants, GLYR1 and GLYR2, and while GLYR2 is known to be localized in plastids, GLYR1 has been reported to be localized in either peroxisomes or the cytosol. Here, we reappraised the intracellular localization of GLYR1 in Arabidopsis thaliana L. Heynh (ecotype Lansberg erecta) using both transiently-transformed suspension cells and stably-transformed plants, in combination with fluorescence microscopy. The results indicate that GLYR1 is localized exclusively to the cytosol regardless of the species, tissue and/or cell type, or exposure of plants to environmental stresses that would increase flux through the GABA pathway. Moreover, the C-terminal tripeptide sequence of GLYR1, -SRE, despite its resemblance to a type 1 peroxisomal targeting signal, is not sufficient for targeting to peroxisomes. Collectively, these results define the cytosol as the intracellular location of GLYR1 and provide not only important insight to the metabolic roles of GLYR1 and the compartmentation of the GABA and photorespiratory pathways in plant cells, but also serve as a useful reference for future studies of proteins proposed to be localized to peroxisomes and/or the cytosol.

  • Detoxification of succinate semialdehyde in Arabidopsis Glyoxylate Reductase and NAD kinase mutants subjected to submergence stress
    Botany, 2012
    Co-Authors: Wendy L. Allan, Gordon J. Hoover, Kevin E. Breitkreuz, Jeffrey C. Waller, Jeffrey P. Simpson, Amanda Rochon, David J. Wolyn, Doris Rentsch, Wayne A. Snedden, Barry J. Shelp
    Abstract:

    Succinate semialdehyde (SSA) is a mitochondrially generated intermediate in the metabolism of γ-aminobutyrate (GABA), which accumulates in response to a variety of biotic and abiotic stresses. SSA can be reduced to γ-hydroxybutyrate (GHB) in plants exposed to various abiotic stress conditions. Recent evidence indicates that distinct cytosolic and plastidial Glyoxylate Reductase isoforms from Arabidopsis thaliana (L.) Heynh (GLYR1 and GLYR2, respectively) catalyze the in vitro conversion of SSA to GHB, as well as Glyoxylate to glycolate, via NADPH-dependent reactions. In the present study, recombinant Arabidopsis GLYR1 was demonstrated to catalyze the NADPH-dependent reduction of both Glyoxylate and SSA simultaneously to glycolate and GHB, respectively. Six-hour time-course experiments with intact vegetative wild-type Arabidopisis plants subjected to submergence demonstrated that GHB accumulates in rosette leaves, and this is accompanied by increasing levels of GABA and alanine, NADH/NAD+ and NADPH/NADP+ r...

  • Research Article Glyoxylate Reductase Isoform 1 is Localized in the Cytosol and Not Peroxisomes in Plant Cells
    2012
    Co-Authors: Steven L. K. Ching, Barry J. Shelp, Amanda Rochon, Satinder K. Gidda, Owen R. Van Cauwenberghe, Robert T. Mullen
    Abstract:

    Glyoxylate Reductase (GLYR) is a key enzyme in plant metabolism which catalyzes the detoxification of both photorespiratory Glyoxylate and succinic semialdehdye, an intermediate of the γ-aminobutyrate (GABA) pathway. Two isoforms of GLYR exist in plants, GLYR1 and GLYR2, and while GLYR2 is known to be localized in plastids, GLYR1 has been reported to be localized in either peroxisomes or the cytosol. Here, we reappraised the intracellular localization of GLYR1 in Arabidopsis thaliana L. Heynh (ecotype Lansberg erecta) using both transiently-transformed suspension cells and stably-transformed plants, in combinationwithfluorescencemicroscopy.TheresultsindicatethatGLYR1islocalizedexclusivelytothe cytosol regardless of the species, tissue and/or cell type, or exposure of plants to environmental stresses that would increase flux through the GABA pathway. Moreover, the C-terminal tripeptide sequence of GLYR1, -SRE, despite its resemblance to a type 1 peroxisomal targeting signal, is not sufficient for targeting to peroxisomes. Collectively, these results define the cytosol as the intracellular location of GLYR1 and provide not only important insight to the metabolic roles of GLYR1 and the compartmentation of the GABA and photorespiratory pathways in plant cells, but also serve as a useful reference for future studies of proteins proposed to be localized to peroxisomes and/or the cytosol.

  • Response of phosphorylated pyridine nucleotides in mature rosette leaves of plants (A) and mature leaves of tobacco plants (B) subjected to cold, heat or submergence
    2011
    Co-Authors: Wendy L. Allan, Shawn M. Clark, Jeffrey P. Simpson, Barry J. Shelp
    Abstract:

    Closed and open circles represent control and experimental plants, respectively. Data represent the mean ±SE; where the bar is not shown, it is within the symbol.Copyright information:Taken from "γ-Hydroxybutyrate accumulation in and tobacco plants is a general response to abiotic stress: putative regulation by redox balance and Glyoxylate Reductase isoforms"Journal of Experimental Botany 2008;59(9):2555-2564.Published online 20 May 2008PMCID:PMC2423657.

Gill Rumsby - One of the best experts on this subject based on the ideXlab platform.

  • Primary cultures of renal proximal tubule cells derived from individuals with primary hyperoxaluria
    Urological Research, 2009
    Co-Authors: Karen L. Price, Sallyanne Hulton, William G. Van’t Hoff, John R. Masters, Gill Rumsby
    Abstract:

    The primary hyperoxalurias, PH1 and PH2, are inherited disorders caused by deficiencies of alanine:Glyoxylate aminotransferase and Glyoxylate Reductase, respectively. Mutations in either of these enzymes leads to endogenous oxalate overproduction primarily in the liver, but most pathological effects are exhibited in the kidney ultimately leading to end-stage renal failure and systemic oxalosis. To provide a non-invasive means of accessing kidney cells from individuals with primary hyperoxaluria, we have derived primary cultures of renal proximal tubule cells from the urine of these patients. The cells stain positively for the epithelial markers pan-cytokeratin and zonula occludens 1 and the proximal tubule marker γ-glutamyl transpeptidase. Mutation analysis confirmed that the cultured cells had the same genotype as the leucocytes of the patients and also expressed Glyoxylate Reductase at the mRNA level, illustrating their potential value as a source of renal material from these individuals.

  • structural basis of substrate specificity in human Glyoxylate Reductase hydroxypyruvate Reductase
    Journal of Molecular Biology, 2006
    Co-Authors: Michael P.s. Booth, Gill Rumsby, R. Conners, Leo R Brady
    Abstract:

    Abstract Human Glyoxylate Reductase/hydroxypyruvate Reductase (GRHPR) is a D -2-hydroxy-acid dehydrogenase that plays a critical role in the removal of the metabolic by-product Glyoxylate from within the liver. Deficiency of this enzyme is the underlying cause of primary hyperoxaluria type 2 (PH2) and leads to increased urinary oxalate levels, formation of kidney stones and renal failure. Here we describe the crystal structure of human GRHPR at 2.2 A resolution. There are four copies of GRHPR in the crystallographic asymmetric unit: in each homodimer, one subunit forms a ternary (enzyme + NADPH + reduced substrate) complex, and the other a binary (enzyme + NADPH) form. The spatial arrangement of the two enzyme domains is the same in binary and ternary forms. This first crystal structure of a true ternary complex of an enzyme from this family demonstrates the relationship of substrate and catalytic residues within the active site, confirming earlier proposals of the mode of substrate binding, stereospecificity and likely catalytic mechanism for these enzymes. GRHPR has an unusual substrate specificity, preferring Glyoxylate and hydroxypyruvate, but not pyruvate. A tryptophan residue (Trp141) from the neighbouring subunit of the dimer is projected into the active site region and appears to contribute to the selectivity for hydroxypyruvate. This first crystal structure of a human GRHPR enzyme also explains the deleterious effects of naturally occurring missense mutations of this enzyme that lead to PH2.

  • Structural basis of substrate specificity in human Glyoxylate Reductase/hydroxypyruvate Reductase.
    Journal of molecular biology, 2006
    Co-Authors: Michael P.s. Booth, Gill Rumsby, R. Conners, R. Leo Brady
    Abstract:

    Abstract Human Glyoxylate Reductase/hydroxypyruvate Reductase (GRHPR) is a D -2-hydroxy-acid dehydrogenase that plays a critical role in the removal of the metabolic by-product Glyoxylate from within the liver. Deficiency of this enzyme is the underlying cause of primary hyperoxaluria type 2 (PH2) and leads to increased urinary oxalate levels, formation of kidney stones and renal failure. Here we describe the crystal structure of human GRHPR at 2.2 A resolution. There are four copies of GRHPR in the crystallographic asymmetric unit: in each homodimer, one subunit forms a ternary (enzyme + NADPH + reduced substrate) complex, and the other a binary (enzyme + NADPH) form. The spatial arrangement of the two enzyme domains is the same in binary and ternary forms. This first crystal structure of a true ternary complex of an enzyme from this family demonstrates the relationship of substrate and catalytic residues within the active site, confirming earlier proposals of the mode of substrate binding, stereospecificity and likely catalytic mechanism for these enzymes. GRHPR has an unusual substrate specificity, preferring Glyoxylate and hydroxypyruvate, but not pyruvate. A tryptophan residue (Trp141) from the neighbouring subunit of the dimer is projected into the active site region and appears to contribute to the selectivity for hydroxypyruvate. This first crystal structure of a human GRHPR enzyme also explains the deleterious effects of naturally occurring missense mutations of this enzyme that lead to PH2.

  • Tissue Differences in the Expression of Mutations and Polymorphisms in the GRHPR Gene and Implications for Diagnosis of Primary Hyperoxaluria Type 2
    Clinical chemistry, 2005
    Co-Authors: Swati Bhat, Emma L. Williams, Gill Rumsby
    Abstract:

    Primary hyperoxaluria type 2 (PH2; OMIM 260000) is an inherited disease of endogenous oxalate overproduction arising from mutations in the GRHPR gene encoding Glyoxylate Reductase. The disease typically presents with urolithiasis or recurrent urinary tract infections and increased urinary oxalate (1). The diagnosis may be supported by l-glyceraciduria, although this does not occur in all cases (2). Definitive diagnosis is currently based on demonstration of diminished Glyoxylate Reductase activity in a liver biopsy (3), although DNA analysis offers a noninvasive method. The GRHPR gene maps to the centromeric region of chromosome 9 (4) and, from Northern blot analysis(5), is ubiquitously expressed, although the bulk of enzyme activity is found in the liver (3)(5). Among the described mutations and polymorphisms (5)(6), c.103delG accounts for 37% of mutant alleles, allowing diagnosis of PH2 to be made by genetic testing (5). One of the polymorphisms, c.579G>A, occurs in exon 6, and the G allele …

  • Molecular analysis of the Glyoxylate Reductase (GRHPR) gene and description of mutations underlying primary hyperoxaluria type 2.
    Human mutation, 2003
    Co-Authors: David P. Cregeen, Sallyanne Hulton, Emma L. Williams, Gill Rumsby
    Abstract:

    Primary hyperoxaluria type 2, an inherited autosomal recessive disorder of endogenous oxalate overproduction, is caused by mutations in the GRHPR gene encoding the Glyoxylate/hydroxypyruvate Reductase enzyme. The GRHPR genes from nineteen unrelated patients with PH2 were analysed for mutations using a combination of PCR-SSCP and sequence analysis of genomic and cDNA. Eleven mutations were identified, seven of which are novel. The mutations included five point mutations: c.84-2A>G, c.295C>T (R99X), c.494G>A (G165D), and c.904C>T (R302C) as well as six minor deletions: c.103delG, c.375delG, c.403_405+2 delAAGT, c.540delT, c.608_609delCT and a more complex mutation in intron 1: c.84-13_c.84-12del; c.84-8_c.84-5del. Aberrant transcripts were demonstrated in hepatic mRNA as a result of the c.403_405+2 delAAGT and c.84-2A>G mutations. In addition, a splice variant lacking 28 bp of exon 1 was expressed in a number of tissues but is of unknown function. Two polymorphisms, c.579A>G in exon 6 and a (CT)(n) microsatellite in intron 8 were identified. Expression studies showed that the G165D and R302C mutants had Glyoxylate Reductase activity 1.5 and 5.6% respectively of the wild type protein. Both mutant proteins were unstable on purification. Although there is wide expression of the GRHPR mRNA demonstrated by northern blot analysis, our study shows that GRHPR protein distribution is predominantly hepatic and concludes that PH2, like the related type 1 disease, is primarily a disorder affecting hepatic Glyoxylate metabolism.

Wendy L. Allan - One of the best experts on this subject based on the ideXlab platform.

  • REVIEW ARTICLE Role of plant Glyoxylate Reductases during stress: a hypothesis
    2016
    Co-Authors: Wendy L. Allan, Shawn M. Clark, Gordon J. Hoover, Barry J. Shelp
    Abstract:

    Molecular modelling suggests that a group of proteins in plants known as the β-hydroxyacid dehydrogenases, or the hydroxyisobutyrate dehydrogenase superfamily, includes enzymes that reduce succinic semialdehyde and Glyoxylate to γ-hydroxybutyrate and glycolate respectively. Recent biochemical and expression studies reveal that NADPH-dependent cytosolic (termed GLYR1) and plastidial (termed GLYR2) isoforms of suc-cinic semialdehyde/Glyoxylate Reductase exist in Arabidopsis. Succinic semialdehyde and Glyoxylate are typically generated in leaves via two distinct metabolic pathways, γ-aminobutyrate and glycolate respectively. In the present review, it is proposed that the GLYRs function in the detoxification of both aldehydes during stress and contribute to redox balance. Outstanding questions are highlighted in a scheme for the subcellular organization of the detoxification mechanism in Arabidopsis. Key words: aldehyde detoxification, Glyoxylate Reductase, β-hydroxyacid dehydrogenase, redox balance, succinic semi

  • Detoxification of succinate semialdehyde in Arabidopsis Glyoxylate Reductase and NAD kinase mutants subjected to submergence stress
    Botany, 2012
    Co-Authors: Wendy L. Allan, Gordon J. Hoover, Kevin E. Breitkreuz, Jeffrey C. Waller, Jeffrey P. Simpson, Amanda Rochon, David J. Wolyn, Doris Rentsch, Wayne A. Snedden, Barry J. Shelp
    Abstract:

    Succinate semialdehyde (SSA) is a mitochondrially generated intermediate in the metabolism of γ-aminobutyrate (GABA), which accumulates in response to a variety of biotic and abiotic stresses. SSA can be reduced to γ-hydroxybutyrate (GHB) in plants exposed to various abiotic stress conditions. Recent evidence indicates that distinct cytosolic and plastidial Glyoxylate Reductase isoforms from Arabidopsis thaliana (L.) Heynh (GLYR1 and GLYR2, respectively) catalyze the in vitro conversion of SSA to GHB, as well as Glyoxylate to glycolate, via NADPH-dependent reactions. In the present study, recombinant Arabidopsis GLYR1 was demonstrated to catalyze the NADPH-dependent reduction of both Glyoxylate and SSA simultaneously to glycolate and GHB, respectively. Six-hour time-course experiments with intact vegetative wild-type Arabidopisis plants subjected to submergence demonstrated that GHB accumulates in rosette leaves, and this is accompanied by increasing levels of GABA and alanine, NADH/NAD+ and NADPH/NADP+ r...

  • Response of phosphorylated pyridine nucleotides in mature rosette leaves of plants (A) and mature leaves of tobacco plants (B) subjected to cold, heat or submergence
    2011
    Co-Authors: Wendy L. Allan, Shawn M. Clark, Jeffrey P. Simpson, Barry J. Shelp
    Abstract:

    Closed and open circles represent control and experimental plants, respectively. Data represent the mean ±SE; where the bar is not shown, it is within the symbol.Copyright information:Taken from "γ-Hydroxybutyrate accumulation in and tobacco plants is a general response to abiotic stress: putative regulation by redox balance and Glyoxylate Reductase isoforms"Journal of Experimental Botany 2008;59(9):2555-2564.Published online 20 May 2008PMCID:PMC2423657.

  • Response of transcripts in mature rosette leaves of plants subjected to salinity, drought, submergence, cold, or heat
    2011
    Co-Authors: Wendy L. Allan, Shawn M. Clark, Jeffrey P. Simpson, Barry J. Shelp
    Abstract:

    Closed and open circles represent control and experimental plants, respectively. Data represent the mean ±SE; where the bar is not shown, it is within the symbol.Copyright information:Taken from "γ-Hydroxybutyrate accumulation in and tobacco plants is a general response to abiotic stress: putative regulation by redox balance and Glyoxylate Reductase isoforms"Journal of Experimental Botany 2008;59(9):2555-2564.Published online 20 May 2008PMCID:PMC2423657.

  • Response of metabolites in mature rosette leaves of plants (A) and mature leaves of tobacco plants (B) subjected to salinity, drought, submergence, cold or heat
    2011
    Co-Authors: Wendy L. Allan, Shawn M. Clark, Jeffrey P. Simpson, Barry J. Shelp
    Abstract:

    Closed and open circles represent control and experimental plants, respectively. Data represent the mean ±SE; where the bar is not shown, it is within the symbol. Note that the drought data only are expressed on a DM basis, rather than a FM basis. TAA represents total amino acids.Copyright information:Taken from "γ-Hydroxybutyrate accumulation in and tobacco plants is a general response to abiotic stress: putative regulation by redox balance and Glyoxylate Reductase isoforms"Journal of Experimental Botany 2008;59(9):2555-2564.Published online 20 May 2008PMCID:PMC2423657.

G. Rumsby - One of the best experts on this subject based on the ideXlab platform.

  • Primary cultures of renal proximal tubule cells derived from individuals with primary hyperoxaluria
    2007
    Co-Authors: Kl Price, Hulton, Masters, G. Rumsby
    Abstract:

    [SA-PO828] Primary Cultures of Renal Proximal Tubule Cells Derived from Individuals with Primary Hyperoxaluria.Karen L. Price, Sally A. Hulton, John R. Masters, Gill Rumsby Nephro-Urology Unit, UCL Institute of Child Health, London, United Kingdom; Department of Nephrology, Birmingham Childrens Hospital, Birmingham, United Kingdom; Institute of Urology, University College London, London, United Kingdom; Clinical Biochemistry, University College London Hospitals, London, United KingdomThe primary hyperoxalurias are inherited disorders caused by deficiencies of two enzymes, alanine:Glyoxylate aminotransferase and Glyoxylate Reductase. Mutations in these enzymes lead to endogenous oxalate overproduction primarily in the liver, but most pathological effects are exhibited in the kidney ultimately leading to end stage renal failure and systemic oxalosis. The variety of presenting features in patients with identical genotypes suggests that as yet unidentified genes influence susceptibility to stone development. To provide a non-invasive means of investigating possible differences in gene expression in renal tubules exposed to oxalate we derived primary cultures of renal proximal tubule cells from the urine of individuals with PH. Urine samples from 17 PH patients have been collected for the isolation of proximal tubule cells. The isolated cells displayed an epithelial morphology, which did not alter on sub-culture, and expressed the epithelial markers pan-cytokeratin and ZO-1. The proximal tubular nature of the isolated cells was confirmed by positive immunocytochemistry for -glutamyl transpeptidase and RT-PCR for the expression of aminopeptidase A. The cells were negative for expression of distal tubular and collecting duct markers, uromodulin and aquaporin 3 respectively. Mutation analysis confirmed that the cultured cells had the same genotype as the leucocytes of the patients and also expressed Glyoxylate Reductase at the mRNA level, illustrating their potential physiological value. Our preliminary results show that primary cultures of human proximal tubule cells can be obtained from urine of patients with PH, providing the means with which to study the impact of these diseases on the kidney.

  • Primary cultures of renal proximal tubule cells derived from individuals with primary hyperoxaluria, in Proceedings of the 8th International Primary Hyperoxaluria Workshop, UCL-Institute of Child Health, 29-30 June 2007, C. J. Danpure & G. Rumsby, ed
    2007
    Co-Authors: Kl Price, Sallyanne Hulton, As Woolf, Jm Masters, G. Rumsby
    Abstract:

    59. Primary cultures of renal proximal tubule cells derived from individuals with primary hyperoxaluriaKaren L Price1, Sally-Anne Hulton2, Adrian S. Woolf1, JohnM. Masters3 and Gill Rumsby41Nephro-Urology Unit, UCL Institute of Child Health, London, UK; 2Department of Nephrology, Birmingham Children's Hospital, Birmingham, UK; 3Prostate Cancer Research Centre, Institute of Urology, University College London, UK; 4Clinical Biochemistry, University College London Hospitals, London, UK. karen.price@ucl.ac.ukThe primary hyperoxalurias (PH1 and PH2) are a group of inherited disorders caused by deficiencies of two enzymes, alanine: Glyoxylate aminotransferase and Glyoxylate Reductase respectively. Mutations in either of these enzymes leads to endogenous oxalate overproduction primarily in the liver, but most pathological effects are exhibited in the kidney ultimately leading to end stage renal failure and systemic oxalosis. The variety of presenting features in patients with identical genotypes suggests that as yet unidentified genes influence susceptibility to stone development, and our goal is to identify such genes.To provide a non-invasive means of investigating possible differences in gene expression in the renal tubules exposed to oxalate we have derived primary cultures of renal proximal tubule cells from the urine of individuals with PH. Urine samples from seventeen PH patients have been collected for the isolation of proximal tubule cells. The isolated cells displayed an epithelial morphology, which did not alter on sub-culture, and expressed the epithelial markers pan-cytokeratin and ZO-1. The proximal tubular nature of the isolated cells was confirmed by positive immunocytochemistry for c-glutamyl transpeptidase and RT-PCR for the expression of aminopeptidase A. Furthermore, the cells were negative for the expression of the distal tubular and collecting duct markers, uromodulin and aquaporin 3 respectively, as assessed by RT-PCR. Mutation analysis confirmed that the cultured cells had the same genotype as the leucocytes of the patients and also expressed Glyoxylate Reductase at the mRNA level, illustrating their potential physiological value.Our preliminary results show that primary cultures of human proximal tubule cells can be obtained from urine of patients with PH, providing the means with which to study the impact of these diseases on the kidney. By defining mechanisms influencing PH development, we may better understand why the disease progresses at different rates in patients with the same genetic abnormality, allowing specific strategies to be devised to slow down or prevent the clinical consequences of the disease.

  • A preliminary account of the properties of recombinant human Glyoxylate Reductase (GRHPR), LDHA and LDHB with Glyoxylate, and their potential roles in its metabolism
    Biochimica et biophysica acta, 2005
    Co-Authors: K. Mdluli, Michael P.s. Booth, R.l. Brady, G. Rumsby
    Abstract:

    Abstract Human lactate dehydrogenase (LDH) is thought to contribute to the oxidation of Glyoxylate to oxalate and thus to the pathogenesis of disorders of endogenous oxalate overproduction. Glyoxylate Reductase (GRHPR) has a potentially protective role metabolising Glyoxylate to the less reactive glycolate. In this paper, the kinetic parameters of recombinant human LDHA, LDHB and GR have been compared with respect to their affinity for Glyoxylate and related substrates. The Km values and specificity constants (Kcat/KM) of purified recombinant human LDHA, LDHB and GRHPR were determined for the reduction of Glyoxylate and hydroxypyruvate. KM values with Glyoxylate were 29.3 mM for LDHA, 9.9 mM for LDHB and 1.0 mM for GRHPR. For the oxidation of Glyoxylate, KM values were 0.18 mM and 0.26 mM for LDHA and LDHB respectively with NAD+ as cofactor. Overall, under the same reaction conditions, the specificity constants suggest there is a fine balance between the reduction and oxidation reactions of these substrates, suggesting that control is most likely dictated by the ambient concentrations of the respective intracellular cofactors. Neither LDHA nor LDHB utilised glycolate as substrate and NADPH was a poor cofactor with a relative activity less than 3% that of NADH. GRHPR had a higher affinity for NADPH than NADH (KM 0.011 mM vs. 2.42 mM). The potential roles of LDH isoforms and GRHPR in oxalate synthesis are discussed.

  • Recent developments in our understanding of primary hyperoxaluria type 2.
    Journal of The American Society of Nephrology, 1999
    Co-Authors: D. P. Cregeen, G. Rumsby
    Abstract:

    Hydroxypyruvate Reductase (HPR) has been partially purified from human liver and can be separated into at least two forms by chromatofocusing; these forms therefore differ in their pI values. Both forms, one with a pI of >7.2 (peak A) and the other with a pI between pH 6.5 and 5.5 (peak B), use NADPH as a cofactor. However, only peak B was able to reduce hydroxypyruvate and Glyoxylate, with a Km of 2.3 mM for the latter substrate. Peak A coeluted with lactate dehydrogenase and could represent lactate dehydrogenase (which is known to reduce hydroxypyruvate) alone or a mixture of proteins with HPR activity. The Km for hydroxypyruvate of the enzyme(s) in peak A (8 mM) was 80 times greater than that of peak B (0.1 mM), suggesting that the HPR enzyme contained in peak B may be more important physiologically, where the hydroxypyruvate concentrations are in the micromolar range. The data presented provide a biochemical explanation for the previously observed differences in the tissue distribution of HPR and Glyoxylate Reductase activities in human subjects and support the claim that diagnoses of primary hyperoxaluria type 2 should be made by measurement of Glyoxylate Reductase activity in the liver.

Shawn M. Clark - One of the best experts on this subject based on the ideXlab platform.

  • REVIEW ARTICLE Role of plant Glyoxylate Reductases during stress: a hypothesis
    2016
    Co-Authors: Wendy L. Allan, Shawn M. Clark, Gordon J. Hoover, Barry J. Shelp
    Abstract:

    Molecular modelling suggests that a group of proteins in plants known as the β-hydroxyacid dehydrogenases, or the hydroxyisobutyrate dehydrogenase superfamily, includes enzymes that reduce succinic semialdehyde and Glyoxylate to γ-hydroxybutyrate and glycolate respectively. Recent biochemical and expression studies reveal that NADPH-dependent cytosolic (termed GLYR1) and plastidial (termed GLYR2) isoforms of suc-cinic semialdehyde/Glyoxylate Reductase exist in Arabidopsis. Succinic semialdehyde and Glyoxylate are typically generated in leaves via two distinct metabolic pathways, γ-aminobutyrate and glycolate respectively. In the present review, it is proposed that the GLYRs function in the detoxification of both aldehydes during stress and contribute to redox balance. Outstanding questions are highlighted in a scheme for the subcellular organization of the detoxification mechanism in Arabidopsis. Key words: aldehyde detoxification, Glyoxylate Reductase, β-hydroxyacid dehydrogenase, redox balance, succinic semi

  • Response of phosphorylated pyridine nucleotides in mature rosette leaves of plants (A) and mature leaves of tobacco plants (B) subjected to cold, heat or submergence
    2011
    Co-Authors: Wendy L. Allan, Shawn M. Clark, Jeffrey P. Simpson, Barry J. Shelp
    Abstract:

    Closed and open circles represent control and experimental plants, respectively. Data represent the mean ±SE; where the bar is not shown, it is within the symbol.Copyright information:Taken from "γ-Hydroxybutyrate accumulation in and tobacco plants is a general response to abiotic stress: putative regulation by redox balance and Glyoxylate Reductase isoforms"Journal of Experimental Botany 2008;59(9):2555-2564.Published online 20 May 2008PMCID:PMC2423657.

  • Response of transcripts in mature rosette leaves of plants subjected to salinity, drought, submergence, cold, or heat
    2011
    Co-Authors: Wendy L. Allan, Shawn M. Clark, Jeffrey P. Simpson, Barry J. Shelp
    Abstract:

    Closed and open circles represent control and experimental plants, respectively. Data represent the mean ±SE; where the bar is not shown, it is within the symbol.Copyright information:Taken from "γ-Hydroxybutyrate accumulation in and tobacco plants is a general response to abiotic stress: putative regulation by redox balance and Glyoxylate Reductase isoforms"Journal of Experimental Botany 2008;59(9):2555-2564.Published online 20 May 2008PMCID:PMC2423657.

  • Response of metabolites in mature rosette leaves of plants (A) and mature leaves of tobacco plants (B) subjected to salinity, drought, submergence, cold or heat
    2011
    Co-Authors: Wendy L. Allan, Shawn M. Clark, Jeffrey P. Simpson, Barry J. Shelp
    Abstract:

    Closed and open circles represent control and experimental plants, respectively. Data represent the mean ±SE; where the bar is not shown, it is within the symbol. Note that the drought data only are expressed on a DM basis, rather than a FM basis. TAA represents total amino acids.Copyright information:Taken from "γ-Hydroxybutyrate accumulation in and tobacco plants is a general response to abiotic stress: putative regulation by redox balance and Glyoxylate Reductase isoforms"Journal of Experimental Botany 2008;59(9):2555-2564.Published online 20 May 2008PMCID:PMC2423657.

  • Expression and purification of recombinant GR2 extracted from cells co-expressing the GroES/GroEL chaperone complex
    2011
    Co-Authors: Jeffrey P. Simpson, Wendy L. Allan, Shawn M. Clark, Gordon J. Hoover, Rosa Di Leo, Preetinder K. Dhanoa, Amina Makhmoudova, Robert T. Mullen, Barry J. Shelp
    Abstract:

    The top row illustrates SDS-PAGE analysis of protein lysates from cells transformed with the truncated gene insert (A) or the empty insert (B), respectively, whereas the bottom row illustrates SDS (C) and immunoblot (D) analysis of protein lysates from cells transformed with the truncated gene insert. The gels were stained with Coomassie Blue, whereas the immunoblot was probed with an anti-His antibody. The EL subunit of the chaperone complex (1) and the recombinant protein (2) are indicated.Copyright information:Taken from "Identification and characterization of a plastid-localized Glyoxylate Reductase isoform: comparison with a cytosolic isoform and implications for cellular redox homeostasis and aldehyde detoxification"Journal of Experimental Botany 2008;59(9):2545-2554.Published online 20 May 2008PMCID:PMC2423656.