Abstract
Anl-amino-acid oxidase (EC 1.4.3.1) that catalyzes the oxidative deamination of twelvel-amino acids has been purified 21-fold and with 14% yield to electrophoretic homogeneity fromChlamydomonas reinhardtii cells by ammonium-sulfate fractionation, gel filtration through Sephacryl and Superose, anion-exchange chromatography and preparative electrophoresis in polyacrylamide gels. The native enzyme is a protein of 470 kDa and consists of eight identical or similarsized subunits of 60 kDa each. Optimum pH and temperature were 8.2 and 55° C, respectively, with a Q10 (45–55° C) of 1.7 and an activation energy of 45 kJ · mol−1. Its absorption spectrum showed, in the visible region, maxima at 360 and 444 nm, characteristic of a flavoprotein with a calculated flavin content of 7.7 mol FAD per mol of native enzyme. ApparentK m values of the twelvel-amino acids which can act as substrates ofl-amino-acid oxidase ranged between 31 μM for phenylalanine and 176 μM for methionine. The effect of several specific group reagents, chelating agents and bivalent cations on enzyme activity has also been studied.
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References
Andrews, P. (1965) The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochem. J.96, 595–606
Blaschko, H., Hope, D.B. (1956) The oxidation ofl-amino acids byMytilus edulis. Biochem. J.62, 335–339
Borchers, R. (1977) Allantoin determination. Anal. Biochem.79, 612–613
Boulanger, P., Osteux, R. (1955a) Activité et spécificité de lal-acidamino-déshydrogénase du foie de dindon. Compt. Rend.241, 125–127
Boulanger, P., Osteux, R. (1955b) Les produits de l'action de lal-acidamino-déshydrogénase du foie de dindon sur l'arginine, l'ornithine et la lysine. Compt. Rend.241, 613–615
Burton, K. (1952) Thel-amino acid oxidase ofNeurospora. Biochem. J.50, 258–268
Duerre, J.A., Chakrabarty, S. (1975)l-Amino acid oxidase ofProteus rettgeri. J. Bacteriol121, 656–663
Fujisawa, S., Hori, K., Miyazawa, K., Ito, K. (1982) Occurrence ofl-amino acid oxidase in the marine red algaGymnogongrus flabelliformis. Bull. Japan. Soc. Sci. Fish.48, 97–103
Gorman, D.S., Levine, R.P. (1965) Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain ofChlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA54, 1665–1669
Harris, E.H. (1989) TheChlamydomonas sourcebook: a compre-hensive guide to Biology and Laboratory use. Academic Press Inc., San Diego
Ito, K., Hori, K., Miyazawa, K. (1987) Purification and some properties ofl-amino acid oxidase fromAmphiroa crassissima Yendo. Hydrobiologia151/152, 563–569
Jovin, T., Chrambach, A., Naughton, M.A. (1964) Apparatus for preparative temperature regulated polyacrylamide gel electrophoresis. Anal. Biochem.9, 351–364
Knight, S.G. (1948) Thel-amino acid oxidase of molds. J. Bacteriol.55, 401–407
Kusakabe, H., Kodama, K., Machida, H., Midorikawa, Y., Kuninaka, A., Misono, H., Soda, K. (1979) Occurrence of a novel enzyme,l-lysine oxidase with antitumor activity in culture extracts ofTrichoderma viride. Agric. Biol. Chem.43, 337–343
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature227, 680–685
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem.193, 265–275
Meyer, R., Pistorius, E.K. (1987) Some properties of Photosystem II preparations fromSynechococcus sp. — presence of anl-amino acid oxidase in Photosystem II complexes fromSynecho-coccus sp. Biochim. Biophys. Acta893, 426–433
Muñoz-Blanco, J., Hidalgo-Martinez, J., Cárdenas, J. (1990) Extracellular deamination ofl-amino acids byChlamydomonas reinhardtii cells. Planta182, 194–198
Nakano, M., Danowski, T.S. (1966) Crystalline mammalianl-amino acid oxidase from rat kidney mitochondria. J. Biol. Chem.241, 2075–2083
Palenik, B., Morel, F.M.M. (1990) Comparison of cell-surfacel-amino acid oxidases from several marine phytoplankton. Mar. Ecol. Prog. Ser.59, 195–201
Paul, J.H., Cooksey, K.E. (1979) Asparagine metabolism and asparaginase activity in a euryhalineChlamydomonas species, Can. J. Microbiol.25, 1443–1451
Pistorius, E.K., Gau, A.E. (1986) Presence of a flavoprotein in O2-evolving Photosystem II preparations from the cyanobacteriumAnacystis nidulans. Biochim. Biophys. Acta849, 203–210
Pistorius, E.K., Voss, H. (1980) Some properties of a basicl-amino-acid oxidase fromAnacystis nidulans. Biochim. Biophys. Acta611, 227–240
Pistorius, E.K., Voss, H. (1982) Presence of an amino acid oxidase in photosystem II ofAnacystis nidulans. Eur. J. Biochem.126, 203–209
Roche, J., Glahn, P.E., Manchon, P., Thoai, N. (1959) Sur une nouvellel-aminoacide-oxydase, activable par le magnesium. Biochim. Biophys. Acta35, 111–122
Siegel, L.M., Monty, K.J. (1966) Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductase. Biochim. Biophys. Acta112, 346–362
Stumpf, P.K., Green, D.E. (1944)l-amino acid oxidase ofProteus vulgaris. J. Biol. Chem.153, 387–399
Ueda, M., Chang, C-C., Ohno, M. (1988) Purification and charac-terization ofl-amino acid oxidase from the venom ofTrimeresurus mucrosquamatus (Taiwan Habu snake). Toxicon26, 695–706
Wellner, D., Meister, A. (1960) Crystallinel-amino amino acid oxidase ofCrotalus adamanteus. J Biol. Chem.235, 2013–2018
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This work was supported by Grant 780-CO2-01 from CICYT, Spain. The skillful secretarial assistance of C. Santos and I. Molina is gratefully acknowledged.
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Piedras, P., Pineda, M., Muñoz, J. et al. Purification and characterization of anl-amino-acid oxidase fromChlamydomonas reinhardtii . Planta 188, 13–18 (1992). https://doi.org/10.1007/BF01160707
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DOI: https://doi.org/10.1007/BF01160707