Abstract
The enzyme which converts 1-aminocyclo-propane-1-carboxylic acid (ACC) into ethylene, ACC oxidase, has been isolated from apple fruits (Malus x domestica Borkh. cv. Golden Delicious), and for the first time stabilized in vitro by 1,10-phenanthroline and purified 170-fold to homogeneity in a five-step procedure. The sodium dodecyl sulfate-denatured and native proteins have similar molecular weights (approx. 40 kDa) indicating that the enzyme is active in its monomeric form. Antibodies raised against a recombinant ACC oxidase over-produced in Escherichia coli from a tomato cDNA recognise the apple-fruit enzyme with high specificity in both crude extracts and purified form. Glycosylation appears to be absent because of (i) the lack of reactivity towards a mixture of seven different biotinylated lectins and (ii) the absence of N-linked substitution at a potential glycosylation site, in a sequenced peptide. Phenylhydrazine and 2-methyl-1-2-dipyridyl propane do not inhibit activity, indicating that ACC oxidase is not a prosthetic-heme iron protein. The partial amino-acid sequence of the native protein has strong homology to the predicted protein of a tomato fruit cDNA demonstrated to encode ACC oxidase.
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Abbreviations
- ACC:
-
1-aminocyclopropane-1-carboxylic acid
- PA:
-
1,10-phenanthroline
- SDS-PAGE:
-
sodium dodeyl sulfatepolyacrylamide gel electrophoresis
References
Apelbaum, A., J.A., Anderson, J.D., Solomos, T., Lieberman, M. (1981) Some characteristics of the system converting 1-ami-nocyclopropane-1-carboxylic acid to ethylene. Plant Physiol. 67, 80–84
Bouzayen, M., Felix, G., Latché, A., Pech, J.C., Boller, T. (1991) Iron: an essential cofactor for the conversion of 1-aminocyclo-propane-1-carboxylic acid to ethylene. Planta 184, 244–247
Bradford M.M. (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
Britsch, L., Grisebach, H. (1986) Purification and characterization of (2S)-flavanone-3-hydroxylase from Petunia hybrida. Eur. J. Biochem. 156, 569–577
Dong, J.G., Olson, D., Silverstone, A., Yang, S.F. (1992) Sequence of a cDNA coding for a 1-aminocyclopropane-1-carboxylate oxidase homolog from apple fruit. Plant Physiol. 98, 1530–1531
Dupille, E., Latché, A., Roques, C., Pech, J.-C. (1992) Stabilisation in vitro et purification de l'enzyme formant l'éthylène chez la pomme. C.R. Acad. Sci. Paris, Série III, t 315, 77–84
Fernandez-Maculet, J.C., Yang, S.F. (1992) Extraction and partial characterization of the ethylene-forming enzyme from apple fruit. Plant Physiol. 99, 751–754
Fritsch, E.F., Maniatis, T., Sambrook, J. (1989) Molecular Cloning: A laboratory manual, 2nd edn., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.J.
Guy, M., Kende, H. (1984) Conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene by isolated vacuoles of Pisum sativum. L., Planta 160, 281–287
Hamilton, A.J., Lycett, G.W., Grierson, D. (1990) Antisense gene that inhibits synthesis of the hormone ethylene in transgenic plants. Nature 346, 284–287
Harlow, E., Lane, D. (1988) Antibodies — A laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Köck, M., Hamilton, A.J., Grierson, D. (1991) eth1, a gene involved in ethylene synthesis in tomato. Plant Mol. Biol. 17, 141–142
Kuai, J., Dilley, D.R. (1992) Extraction, partial purification and characterization of 1-aminocyclopropane-1-carboxylic acid oxidase from apple fruit. Postharvest Biol. Technol. 1, 203–211
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227, 680–685
Martinat, N., Huet, J.C., Nespoulous, C., Combarnous, Y., Per-nollet, J.C. (1991) Determination of the primary and secondary structures of the dromedary (Camelus dromedarius) prolactin and comparison with prolactins from other species. Biochim. Biophys. Acta 1077, 339–345
McGarvey, J., Yu, H., Christoffersen, R.E. (1990) Nucleotide sequence of a ripening-related cDNA from avocado fruit. Plant Mol. Biol. 15, 165–167
McGarvey, J., Christoffersen, R.E. (1992) Characterization and kinetic parameters of ethylene-forming enzyme from avocado fruit. J. Biol. Chem. 267, 5964–5967
Meyer, Y., Grosset, J., Chartier, Y., Cleyet-Marel, J.C. (1988) Preparation by two-dimensional electrophoresis of proteins for antibody production: Antibodies against proteins whose synthesis is reduced by auxin in tobacco mesophyll protoplasts. Electrophoresis 9, 704–712
Mitchell, T., Porter, A.J.R., John, P. (1988) Authentic activity of the ethylene-forming enzyme observed in membranes obtained from kiwi fruit Actinidia deliciosa. New Phytol. 109, 313–319
Ortiz de Montellano, P.R., Reich, N.O. (1986). Inhibition of cytochrome P-450 enzymes. In: Cytochrome P-450, structure, mechanism, and biochemistry, (pp. 273–314, P.R. Ortiz de Montellano, ed). Plenum Press, New York
Ross, G.S., Knighton, M.L., Lee, M.L. (1992) An ethylene-related cDNA from ripening apples. Plant Mol. Biol. 19, 231–238
Smith, V.A., Gaskin, P., MacMillan, J. (1990) Partial purification and characterization of the gibberellin A20 3β-hydroxylase from seeds of Phaseolus vulgaris. Plant Physiol. 94, 1390–1401
Smith, J.J., Ververidis, P., John, P. (1992) Characterisation of the ethylene-forming enzyme partially purified from melon. Phytochemistry 31, 1485–1494
Spanu, P., Reinhardt, D., Boller, T. (1991) Analysis and cloning of the ethylene-forming enzyme from tomato by functional expression of its mRNA in Xenopus laevis oocytes. EMBO J. 10, 2007–2013
Tabor, S. (1990) Expression using the T7 RNA polymerase/ promoter system. In: Current protocols in molecular biology Aubusel, F.A., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, Y.A., Struhl, K., eds. pp 16.2.1–16.2.11, Green Publishing, Wiley Intersiences New York
Towbin, H., Staehelin, T., Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedures and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354
Ververidis, P., John, P. (1991) Complete recovery in vitro of ethylene-forming enzyme activity. Phytochemistry 30, 725–727
Wang, H., Woodson, W.R. (1991) A flower senescence-related mRNA from carnation shares sequence similarity with fruit ripening-related mRNAs involved in ethylene biosynthesis. Plant. Physiol. 96, 1000–1001
Yang, S.F., Hoffman, N.E. (1984) Ethylene biosynthesis and its regulation in higher plants. Annu. Rev. Plant Physiol. 35, 155–189
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Authors are grateful to Professor Grierson and Dr. Hamilton (University of Nottingham, UK) for providing us with pTOM 13 clone, to Dr. Tabor (Harvard Medical School, Boston, Mass., USA) for the generous gift of plasmid pT7-7 and E. coli strain K38/pGPl-2, to Gisèle Borderie (Centre de Physiologie Végétale, Université Paul Sabatier, Toulouse, France) for amino-acid sequencing, to Bernard Meunier (Chimie de Coordination, CNRS, Toulouse, France) for helpful suggestions and to Dr. John Bull (recipient of a postdoctoral fellowship from the French Ministry of Education, ENSAT, Toulouse, France) for critical reviewing of the manuscript. This work was supported by the Ministère de l'Education Nationale (Jeune Equipe), the EEC (ECLAIR Grant AGRE 015) and the NATO (grant 383/88).
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Dupille, E., Rombaldi, C., Lelièvre, JM. et al. Purification, properties and partial amino-acid sequence of 1-aminocyclopropane-1-carboxylic acid oxidase from apple fruits. Planta 190, 65–70 (1993). https://doi.org/10.1007/BF00195676
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DOI: https://doi.org/10.1007/BF00195676