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Measurement of Enzyme Activities

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Photorespiration

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1653))

Abstract

The determination of enzyme activities in organ or organellar extracts is an important means of investigating metabolic networks and allows testing the success of enzyme-targeted genetic engineering. It also delivers information on intrinsic enzyme parameters such as kinetic properties or impact of effector molecules. This chapter provides protocols on how to assess activities of the enzymes of the core photorespiratory pathway, from 2-phosphoglycolate phosphatase to glycerate 3-kinase.

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References

  1. Bauwe H, Hagemann M, Fernie AR (2010) Photorespiration: players, partners and origin. Trends Plant Sci 15:330–336

    Article  CAS  PubMed  Google Scholar 

  2. Gibon Y, Blaesing OE, Hannemann J, Carillo P, Höhne M, Hendriks JHM, Palacios N, Cross J, Selbig J, Stitt M (2004) A robot-based platform to measure multiple enzyme activities in arabidopsis using a set of cycling assays: comparison of changes of enzyme activities and transcript levels during diurnal cycles and in prolonged darkness. Plant Cell 16:3304–3325

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  4. Bourguignon J, Neuburger M, Douce R (1988) Resolution and characterization of the glycine cleavage reaction in pea leaf mitochondria. Properties of the forward reaction catalysed by glycine decarboxylase and serine hydroxymethyltransferase. Biochem J 255:169–178

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Keech O, Dizengremel P, Gardeström P (2005) Preparation of leaf mitochondria from Arabidopsis thaliana. Physiol Plant 124:403–409

    Article  CAS  Google Scholar 

  6. Seifried A, Knobloch G, Duraphe PS, Segerer G, Manhard J, Schindelin H, Schultz J, Gohla A (2014) Evolutionary and structural analyses of mammalian haloacid dehalogenase-type phosphatases AUM and chronophin provide insight into the basis of their different substrate specificities. J Biol Chem 289:3416–3431

    Article  CAS  PubMed  Google Scholar 

  7. Richardson KE, Tolbert NE (1961) Phosphoglycolic acid phosphatase. J Biol Chem 236:1285–1290

    CAS  PubMed  Google Scholar 

  8. Allen KN, Dunaway-Mariano D (2009) Markers of fitness in a successful enzyme superfamily. Curr Opin Struct Biol 19:658–665

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Hardy P, Baldy P (1986) Corn phosphoglycolate phosphatase—purification and properties. Planta 168:245–252

    CAS  PubMed  Google Scholar 

  10. Schwarte S, Bauwe H (2007) Identification of the photorespiratory 2-phosphoglycolate phosphatase, PGLP1, in Arabidopsis. Plant Physiol 144:1580–1586

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Somerville CR, Ogren WL (1979) A phosphoglycolate phosphatase-deficient mutant of Arabidopsis. Nature 280:833–836

    Article  CAS  Google Scholar 

  12. Clagett CO, Tolbert NE, Burris RH (1949) Oxidation of α-hydroxy acids by enzymes from plants. J Biol Chem 178:977–987

    CAS  PubMed  Google Scholar 

  13. Dellero Y, Mauve C, Boex-Fontvieille E, Flesch V, Jossier M, Tcherkez G, Hodges M (2015) Experimental evidence for a hydride transfer mechanism in plant glycolate oxidase catalysis. J Biol Chem 290:1689–1698

    Article  PubMed  Google Scholar 

  14. Pennati A, Gadda G (2011) Stabilization of an intermediate in the oxidative half-reaction of human liver glycolate oxidase. Biochemistry 50:1–3

    Article  CAS  PubMed  Google Scholar 

  15. Hackenberg C, Kern R, Hüge J, Stal LJ, Tsuji Y, Kopka J, Shiraiwa Y, Bauwe H, Hagemann M (2011) Cyanobacterial lactate oxidases serve as essential partners in N2 fixation and evolved into photorespiratory glycolate oxidases in plants. Plant Cell 23:2978–2990

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Yamaguchi K, Nishimura M (2000) Reduction to below threshold levels of glycolate oxidase activities in transgenic tobacco enhances photoinhibition during irradiation. Plant Cell Physiol 41:1397–1406

    Article  CAS  PubMed  Google Scholar 

  17. Feierabend J, Beevers H (1972) Developmental studies on microbodies in wheat leaves: I. Conditions influencing enzyme development. Plant Physiol 49:28–32

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Macheroux P, Massey V, Thiele DJ, Volokita M (1991) Expression of spinach glycolate oxidase in Saccharomyces cerevisiae: purification and characterization. Biochemistry 30:4612–4619

    Article  CAS  PubMed  Google Scholar 

  19. Kisaki T, Tolbert NE (1969) Glycolate and glyoxylate metabolism by isolated peroxisomes or chloroplasts. Plant Physiol 44:242–250

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kisaki T, Tolbert NE (1970) Glycine as substrate for photorespiration. Plant Cell Physiol 11:247–258

    Article  CAS  Google Scholar 

  21. Hirotsu K, Goto M, Okamoto A, Miyahara I (2005) Dual substrate recognition of aminotransferases. Chem Rec 5:160–172

    Article  CAS  PubMed  Google Scholar 

  22. Dunathan HC (1966) Conformation and reaction specificity in pyridoxal phosphate enzymes. Proc Natl Acad Sci U S A 55:712–716

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Igarashi D, Tsuchida H, Miyao M, Ohsumi C (2006) Glutamate:glyoxylate aminotransferase modulates amino acid content during photorespiration. Plant Physiol 142:901–910

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Liepman AH, Olsen LJ (2001) Peroxisomal alanine:glyoxylate aminotransferase (AGT1) is a photorespiratory enzyme with multiple substrates in Arabidopsis thaliana. Plant J 25:487–498

    Article  CAS  PubMed  Google Scholar 

  25. Stafford HA, Magaldi A, Vennesland B (1954) The enzymatic reduction of hydroxypyruvic acid to D-glyceric acid in higher plants. J Biol Chem 207:621–629

    CAS  PubMed  Google Scholar 

  26. Tolbert NE, Yamazaki RK, Oeser A (1970) Localization and properties of hydroxypyruvate and glyoxylate reductases in spinach leaf particles. J Biol Chem 245:5129–5136

    CAS  PubMed  Google Scholar 

  27. Zelitch I (1953) Oxidation and reduction of glycolic and glyoxylic acids in plants: II. Glyoxylic acid reductase. J Biol Chem 201:719–726

    CAS  PubMed  Google Scholar 

  28. Zelitch I (1955) The isolation and action of crystalline glyoxylic acid reductase from tobacco leaves. J Biol Chem 216:553–575

    CAS  PubMed  Google Scholar 

  29. Timm S, Nunes-Nesi A, Pärnik T, Morgenthal K, Wienkoop S, Keerberg O, Weckwerth W, Kleczkowski LA, Fernie AR, Bauwe H (2008) A cytosolic pathway for the conversion of hydroxypyruvate to glycerate during photorespiration in Arabidopsis. Plant Cell 20:2848–2859

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Allan WL, Clark SM, Hoover GJ, Shelp BJ (2009) Role of plant glyoxylate reductases during stress: a hypothesis. Biochem J 423:15–22

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Bartsch O, Hagemann M, Bauwe H (2008) Only plant-type (GLYK) glycerate kinases produce D-glycerate 3-phosphate. FEBS Lett 582:3025–3028

    Article  CAS  PubMed  Google Scholar 

  32. Kern R, Bauwe H, Hagemann M (2011) Evolution of enzymes involved in the photorespiratory 2-phosphoglycolate cycle from cyanobacteria via algae toward plants. Photosynth Res 109:103–114

    Article  CAS  PubMed  Google Scholar 

  33. Kleczkowski LA, Randall DD, Zahler WL (1985) The substrate specificity, kinetics, and mechanism of glycerate-3-kinase from spinach leaves. Arch Biochem Biophys 236:185–194

    Article  CAS  PubMed  Google Scholar 

  34. Kleczkowski LA, Randall DD (1985) Light and thiol activation of maize leaf glycerate kinase—the stimulating effect of reduced thioredoxins and ATP. Plant Physiol 79:274–277

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Bartsch O, Mikkat S, Hagemann M, Bauwe H (2010) An autoinhibitory domain confers redox regulation to maize glycerate kinase. Plant Physiol 153:832–840

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Kikuchi G, Motokawa Y, Yoshida T, Hiraga K (2008) Glycine cleavage system: reaction mechanism, physiological significance, and hyperglycinemia. Proc Jpn Acad Ser B Phys Biol Sci 84:246–263

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Sagers RD, Gunsalus IC (1961) Intermediary metabolism of Diplococcus glycinophilus: I. Glycine cleavage and one-carbon interconversion. J Bacteriol 81:541–549

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Besson V, Rebeille F, Neuburger M, Douce R, Cossins EA (1993) Effects of tetrahydrofolate polyglutamates on the kinetic parameters of serine hydroxymethyltransferase and glycine decarboxylase from pea leaf mitochondria. Biochem J 292:425–430

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Klein SM, Sagers RD (1966) Glycine metabolism: I. Properties of the system catalyzing the exchange of bicarbonate with the carboxyl-group of glycine in Peptococcus glycinophilus. J Biol Chem 241:197–205

    CAS  PubMed  Google Scholar 

  40. Hasse D, Mikkat S, Hagemann M, Bauwe H (2009) Alternative splicing produces an H-protein with better substrate properties for the P-protein of glycine decarboxylase. FEBS J 276:6985–6991

    Article  CAS  PubMed  Google Scholar 

  41. Yan L-J, Thangthaeng N, Forster MJ (2008) Changes in dihydrolipoamide dehydrogenase expression and activity during postnatal development and aging in the rat brain. Mech Ageing Dev 129:282–290

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Schirch V, Szebenyi DM (2005) Serine hydroxymethyltransferase revisited. Curr Opin Chem Biol 9:482–487

    Article  CAS  PubMed  Google Scholar 

  43. Rebeille F, Neuburger M, Douce R (1994) Interaction between glycine decarboxylase, serine hydroxymethyltransferase and tetrahydrofolate polyglutamates in pea leaf mitochondria. Biochem J 302:223–228

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. McClung CR, Hsu M, Painter JE, Gagne JM, Karlsberg SD, Salome PA (2000) Integrated temporal regulation of the photorespiratory pathway. Circadian regulation of two Arabidopsis genes encoding serine hydroxymethyltransferase. Plant Physiol 123:381–392

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Voll LM, Jamai A, Renné P, Voll H, McClung CR, Weber APM (2006) The photorespiratory Arabidopsis shm1 mutant is deficient in SHM1. Plant Physiol 140:59–66

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Engel N, Ewald R, Gupta KJ, Zrenner R, Hagemann M, Bauwe H (2011) The presequence of Arabidopsis serine hydroxymethyltransferase SHM2 selectively prevents import into mesophyll mitochondria. Plant Physiol 157:1711–1720

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Sopitthummakhun K, Maenpuen S, Yuthavong Y, Leartsakulpanich U, Chaiyen P (2009) Serine hydroxymethyltransferase from Plasmodium vivax is different in substrate specificity from its homologues. FEBS J 276:4023–4036

    Article  CAS  PubMed  Google Scholar 

  48. Zhang Y, Sun K, Roje S (2008) An HPLC-based fluorometric assay for serine hydroxymethyltransferase. Anal Biochem 375:367–369

    Article  CAS  PubMed  Google Scholar 

  49. Taylor RT, Weissbach H (1965) Radioactive assay for serine transhydroxymethylase. Anal Biochem 13:80–84

    Article  CAS  Google Scholar 

  50. Eisenhut M, Kahlon S, Hasse D, Ewald R, Lieman-Hurwitz J, Ogawa T, Ruth W, Bauwe H, Kaplan A, Hagemann M (2006) The plant-like C2 glycolate pathway and the bacterial-like glycerate cycle cooperate in phosphoglycolate metabolism in cyanobacteria. Plant Physiol 142:333–342

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Uyeda K, Rabinowitz JC (1963) Fluorescence properties of tetrahydrofolate and related compounds. Anal Biochem 6:100–108

    Article  CAS  PubMed  Google Scholar 

  52. Boyes DC, Zayed AM, Ascenzi R, McCaskill AJ, Hoffman NE, Davis KR, Gorlach J (2001) Growth stage-based phenotypic analysis of Arabidopsis: a model for high throughput functional genomics in plants. Plant Cell 13:1499–1510

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Timm S, Bauwe H (2013) The variety of photorespiratory phenotypes – employing the current status for future research directions on photorespiration. Plant Biol (Stuttg) 15:737–747

    Article  CAS  Google Scholar 

  54. Delieu T, Walker DA (1972) An improved cathode for the measurement of photosynthetic oxygen evolution by isolated chloroplasts. New Phytol 71:201–225

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by grants of the Deutsche Forschungsgemeinschaft particularly in the DFG Forschergruppe 1186 Photorespiration: Origin and Metabolic Integration in Interacting Compartments (Promics). Stefan Timm (PGLP and optical tests), Maria Wittmiß (GCS), and Ralph Ewald (SHMT), all at Rostock University, kindly double-checked the provided protocols.

Author information

Authors and Affiliations

  1. Plant Physiology Department, University of Rostock, 18059, Rostock, Germany

    Hermann Bauwe

Authors
  1. Hermann Bauwe
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Correspondence to Hermann Bauwe .

Editor information

Editors and Affiliations

  1. Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany

    Alisdair R. Fernie

  2. Plant Physiology Department, University of Rostock, Rostock, Germany

    Hermann Bauwe

  3. Institute of Plant Biochemistry, Cluster of Excellence on Plant Sciences (CEPLAS), Heinrich-Heine-University, Düsseldorf, Germany

    Andreas P.M. Weber

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Bauwe, H. (2017). Measurement of Enzyme Activities. In: Fernie, A., Bauwe, H., Weber, A. (eds) Photorespiration. Methods in Molecular Biology, vol 1653. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7225-8_3

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  • DOI: https://doi.org/10.1007/978-1-4939-7225-8_3

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7224-1

  • Online ISBN: 978-1-4939-7225-8

  • eBook Packages: Springer Protocols

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