SEARCH

SEARCH BY CITATION

Keywords:

  • galactose-1-phosphate uridlytransferase;
  • galactose metabolism;
  • Leloir pathway;
  • Rhodophyta;
  • Galdieria sulphuraria

Abstract

  1. Top of page
  2. Abstract
  3. References

The galactose-1-phosphate uridyltransferase of the red alga Galdieria sulphuraria has been purified about 1800-fold to a final specific activity of approximately 140 U/mg protein. The purification involved chromatography on DEAE-Fractogel, hydroxyapatite, decyl-agarose, and DEAE-Tentacle gel. After SDS/PAGE, the enzyme preparation showed only one protein band of 42 kDa. The enzyme is a homodimer with a molecular mass of 82 kDa as estimated from the sedimentation velocity or 60 kDa as estimated by gel filtration. It has a broad pH optimum between pH 7 and pH 9. The apparent Km values for the forward and backward reactions are Km(Glc1P) = 105 μM, Km(UDP-galactose) = 30 μM, Km(Gal1P) = 400 μM, and Km(UDP-Glc) = 20 μM. The activation energy of the reaction is 45 kJ mol-1. The enzyme is specific for the galactose 1-phosphate to UDP-galactose interconversion in the Leloir pathway while the alternate enzyme for the Isselbacher pathway, UDP-galactose pyrophosphorylase, could not be detected in G. sulphuraria.

Enzymes
 

Galactose-1-phosphate uridyltransferase (EC 2.7.7.12)

 

UDP-galactose pyrophosphorylase (EC 2.7.7.10)

 

UDP-glucose pyrophosphorylase (EC 2.7.7.9)

References

  1. Top of page
  2. Abstract
  3. References
  • Banroques, J., Gregori, C. & Schapira, F. (1981) Purification and characterization of human erythrocyte uridylyltransferase, Biochim. Biophys. Acta 657, 374382.
  • Bergmeyer, H. U. (1983) Methods of enzymatic analysis, Verlag Chemie, Weinheim.
  • Bertoli, D. & Segal, S. (1966) Developmental aspects and some characteristics of mammalian galactose 1-phosphate uridyltransferase, J. Biol. Chem. 241, 40234029.
  • Blum, H., Beier, H. & Gross, H. J. (1987) Improved silver staining for plant proteins, RNA, and DNA in polyacrylamide gels, Electrophoresis 8, 9399.
  • Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding, Anal. Biochem. 72, 248254.
  • Dressler, K., Bliedlingmaier, S., Grossberger, H., Kemmner, J., Nölle, U., Rodmanis-Blumer, A. & Hess, D. (1982) Galactose metabolism in Petunia, Z. Pflanzenphysiol. 107, 409418.
  • Ford, T. W. (1979) Ribulose 1,5-bisphosphate carboxyiase from the thermophilic, acidophilic alga Cyanidium caldarium Geitler. Purification, characterisation and thermostability of the enzyme, Biochim. Biophys. Acta 569, 239248.
  • Göring, H. & Reckin, E. (1968) Einfluß von d-Galaktose auf den Kohlenhydratstoffwechsel pflanzlicher Gewebe, Flora 159, 82103.
  • Gross, K. C. & Pharr, D. M. (1982) A potential pathway for galactose metabolism in Cucumis sativus L., a stachyose transporting species, Plant Physiol. (Bethesda) 69, 117121.
  • Gross, W. & Schnarrenberger, C. (1995) Heterotrophic growth of two strains of the acido-thermophilic red alga Galdieria sulphuraria, Plant Cell Physiol 36, 633638.
  • Helmer, G. R. J. & Williams, V. P. (1981) Purification of galactose 1-phosphate uridylyltransferase from human placenta. Arch. Biochem. Biophys. 210, 573580.
  • Inouhe, M., Yamamoto, R. & Masuda, Y. (1987) Effects of indolacetic acid and galactose on the UTP level and UDP-glucose formation in Avena coleoptile and Vigna epicotyl segments, Physiol. Plant. 69, 579585.
  • Isselbacher, K. J. (1958) A mammalian uridinediphosphate galactose pyrophosphorylase, J. Biol. Chem. 232, 429444.
  • Laemmli, U. K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4, Nature 227, 680685.
  • Loewus, F. A. & Tanner, W. (1982) Plant carbohydrates I, Intracellular carbohydrates, in Encyclopedia of Plant Physiology, vol. 13A, Springer-Verlag, Berlin .
  • Loughman, B. C., Ratcliffe, R. G. & Schwabe, J. W. R. (1989) Galactose metabolism in Zea mays root tissues observed by 31P-NMR spectroscopy, Plant Sci. 59, 1123.
  • Lück, H. (1957) Catalase, in Methods in enzymatic analysis (Bergmeyer, H. U., ed.) pp. 885894, Academic Press, New York .
  • Maretzki, A. & Thom, M. (1978) Characteristics of a galactose-adapted sugarcane cell line grown in suspension culture, Plant Physiol. (Bethesda) 61, 544548.
  • Martin, R. G. & Ames, B. N. (1961) A method for determining the sedimentation behavior of enzymes: application to protein mixtures, J. Biol. Chem. 236, 13721379.
  • Mayes, J. S. (1976) Purification, properties, and isozyme pattern of galactose 1-phosphate uridyltransferase from calf liver, Arch. Biochem. Biophys. 172, 715720.
  • Pazur, J. H. & Shadaksharaswamy, M. (1961) Galactose 1-phosphate uridyl transferase activity in soybean extracts, Biochem. Biophys. Res. Commun. 5, 130134.
  • Roberts, R. M., Heishman, A. & Wicklin, C. (1971) Growth inhibition and metabolite pool levels in plant tissues fed d-glucosamine and d-galactose, Plant Physiol. (Bethesda) 48, 3642.
  • Saito, S., Ozutsumi, M. & Kurahashi, K. (1967) Galactose 1-phosphate uridylyltransferase of Escherichia coll. II. Further purification and characterization, J. Biol. Chem. 242, 23622368.
  • Schnarrenberger, C., Oeser, A. & Tolbert, N. E. (1973) Two isoenzymes each of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in spinach leaves, Arch. Biochem. Biophys. 154, 438448.
  • Segawa, T. & Fukasawa, T (1979) The enzymes of the galactose cluster in Saccharomyces cerevisiae. Purification and characterization of galactose 1-phosphate uridylyltransferase, J. Biol. Chem. 254, 1070710709.
  • Smart, E. L. & Pharr, D. M. (1981) Separation and characteristics of galactose 1-phosphate and glucose-1-phosphate uridyltransferase from fruit peduncles of cucumber, Planta (Heidelb.) 153, 370375.
  • Warburg, O. & Christian, W. (1941) Isolierung und Kristallisation des Gärungsferments Enolase, Biochem Z. 310, 384421.
  • Williams, V. P. (1978) Purification and some properties of galactose 1-phosphate uridyltransferase from human red cells, Arch. Biochem. Biophys. 191, 182191.
  • Williams, V. P. (1978) Purification and some properties of galactose 1-phosphate uridyltransferase from human red cells, Arch. Biochem. Biophys. 191, 182191.
  • Wong, L. & Frey, P. A. (1974) Galactose-1-phosphate uridylyltransferase: rate studies confirming a uridylyl-enzyme intermediate on the catalytic pathway, Biochemistry 13, 38893894.
  • Yamamoto, R., Inouhe, M. & Masuda, Y. (1988) Galactose inhibition of auxin-induced growth of mono- and dicotyledonous plants, Plant Physiol (Bethesda) 86, 12231227.