Abstract
1-Aminocyclopropane-l-carboxylic acid (ACC) synthase (EC 4.4.1.14) was purified 5000-fold from LiCl-induced tomato fruit slices by conventional and high-performance liquid chromatography. In the final preparation the enzyme is estimated 50% pure. Two-dimensional gel electrophoresis indicates that ACC synthase activity is associated with a 45-kD polypeptide.
Due to the extremely low abundance of the protein it was necessary to scale up the extraction in order to obtain reasonable amounts for sequence analysis. Therefore, 200 kg of tomatoes were extracted on semi-industrial scale and purified as before. Using the electroblotting method developed by Vandekerckhove et al. (1985), we obtained sequence data from the N-terminus of the protein. Internal peptide sequences were determined after tryptic digestion and separation of the peptides by reversed-phase chromatography.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
Abeles FB, 1985. Ethylene and plant development: an introduction. In: Roberts JA and Tucker GA. Ethylene and plant development. Butterworths, London.
Acaster MA and Kende H, 1983. Properties and partial purification of 1- aminocyclopropane-l-carboxylate synthase. Plant Physiol. 72: 139–145.
Bauw G, De Loose M, Inzé D, Van Montagu M and Vandekerckhove J, 1987. Alterations in the phenotype of plant cells studied by NH2-terminal amino acid-sequence analysis of proteins electroblotted from two-dimensional gel-separated total extracts. Proc Natl Acad Sci USA 84: 4806–4810.
Bleecker AB, Kenyon WH, Somerville SC and Kende H, 1986. Use of monoclonal antibodies in the purification and characterization of 1-aminocyclopropane1-carboxylate synthase, an enzyme in ethylene biosynthesis. Proc Natl Acad Sci USA 83: 7755–7759.
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.
Brady CJ, 1987. Fruit ripening. Ann Rev Plant Physiol 38: 155–178.
Ecker JR and Davis RW, 1987. Plant defense genes are regulated by ethylene. Proc Natl Acad Sci USA 84: 5202–5206.
Kende H, Bleecker AB, Kenyon WH and Mayne RG, 1986. Enzymes of ethylene biosynthesis. In: Bopp M. Plant growth substances 1985. Springer-Verlag, Berlin.
Laemmli UK, 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.
Lincoln JE, Cordes S, Read E and Fischer RI, 1987. Regulation of gene expression by ethylene during Lycopersicon esculentum(tomato) fruit development. Proc Natl Acad Sci USA 84: 2793–2797.
Lizada MCC and Yang SF, 1979. A simple and sensitive assay for 1-aminocyclopropane-l-carboxylic acid. Anal Biochem 100: 140–145.
Nielsen BL and Brown LR, 1984. The basis for colored silver-protein complex formation in stained polyacrylamide gels. Anal Biochem 141: 311–315.
Reddy VA and Rao NA, 1976. Dihydrofolate reductase from soybean seedlings. Characterization of the enzyme purified by affinity chromatography. Arch Biochem Biophys 174: 675–683.
Slater A, Maunders MJ, Edwards K, Schuch W and Grierson D, 1985. Isolation and characterisation of cDNA clones from tomato polygalacturonase and other ripening-related proteins. Plant Mol Biol 5: 137–147.
United States Department of Agriculture, 1976. United States standards for grades of fresh tomatoes. Agricultural Marketing Service. USDA, Washington.
Vandekerckhove J, Bauw G, Puype M, Van Damme J and Van Montagu M, 1985. Protein-blotting on Polybrene-coated glass-fiber sheets. A basis for acid hydrolysis and gas-phase sequencing of picomole quantities of protein previously separated on sodium dodecyl sulfate/polyacrylamide gel. Eur J Biochem 152: 9–19.
Yang SF and Hoffman NE, 1984. Ethylene biosynthesis and its regulation in higher plants. Ann Rev Plant Physiol 35: 155–189.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1989 Kluwer Academic Publishers
About this chapter
Cite this chapter
Van Der Straeten, D., Van Wiemeersch, L., Van Damme, J., Goodman, H., Montagu, M.V. (1989). Purification and Amino-Acid Sequence Analysis of 1-Aminocyclopropane-1-Carboxylic Acid Synthase from Tomato Pericarp. In: Clijsters, H., De Proft, M., Marcelle, R., Van Poucke, M. (eds) Biochemical and Physiological Aspects of Ethylene Production in Lower and Higher Plants. Advances in Agricultural Biotechnology, vol 26. Springer, Dordrecht. https://doi.org/10.1007/978-94-009-1271-7_11
Download citation
DOI: https://doi.org/10.1007/978-94-009-1271-7_11
Publisher Name: Springer, Dordrecht
Print ISBN: 978-94-010-7065-2
Online ISBN: 978-94-009-1271-7
eBook Packages: Springer Book Archive