Purification of extensin from cell walls of tomato (hybrid of Lycopersicon esculentum and L. peruvianum) cells in suspension culture

• Published in: Planta 1993, Vol.191 (4), p.457-469
• Main Authors: Brownleader, M.D. (London Univ., Egham, Surrey (United Kingdom). Dept. of Biochemistry), Dey, P.M
• Format: Article
• Language: English
• Subjects: Agronomy. Soil science and plant productions; Amino Acid Sequence; Amino Acids - analysis; Arthybride; Biological and medical sciences; Biotechnology; Cell biochemistry; CELL CULTURE; Cell physiology; Cell Wall - chemistry; CELL WALLS; Cells, Cultured; CULTIVO DE CELULAS; CULTURE DE CELLULE; Cultured cells; Extensin; Fundamental and applied biological sciences. Psychology; Gels; GLICOPROTEINAS; Glycoprotein; GLYCOPROTEINE; GLYCOPROTEINS; Glycoproteins - chemistry; Glycoproteins - isolation & purification; Glycosylation; HIBRIDOS; HYBRIDE; HYBRIDS; Hydroxyproline - analysis; LYCOPERSICON; Molecular Conformation; Molecular Sequence Data; Molecular weight; Monomers; PARED CELULAR; PAROI CELLULAIRE; PEROXIDASAS; Peroxidase; PEROXIDASES; Peroxidases - metabolism; PEROXYDASE; Plant cells; Plant physiology and development; Plant Proteins - chemistry; Plant Proteins - isolation & purification; Plants; Protein precursors; Protein Precursors - metabolism; Proteins; Space life sciences; Tomate; Vegetables - chemistry; Zellkultur; Zellwand
• ISSN: 0032-0935; 1432-2048
• DOI: 10.1007/BF00195747

Extensin, a hydroxyproline-rich glycoprotein comprising substantial amounts of β-L-arabinose-hydroxyproline glycosidic linkages is believed to be insolubilized in the cell wall during host-pathogen interaction by a peroxidase/hydroperoxide-mediated cross-linking process. Both extensin precursor and extensin peroxidase were ionically eluted from intact water-washed tomato (hybrid of Lycopersicon esculentum Mill. and L. peruvianum L. (Mill.) cells in suspension cultures and purified to homogeneity by a rapid and simple procedure under mild and non-destructive experimental conditions. The molecular weight of native extensin precursor was estimated to be greater than 240—300 kDa by Superose-12 gel-filtration chromatography. Extensin monomers have previously been designated a molecular weight of approximately 80 kDa. Our results indicate that salt-eluted extensin precursor is not monomeric. Agarose-gel electrophoresis, Superose-12-gel-filtration, extensin-peroxidase-catalysed cross-linking, Mono-S ion-exchange fast protein liquid chromatography (FPLC), and peptide-sequencing data confirmed the homogeneity of the extensin preparation. Evidence that the purified protein was extensin is attributed to the presence of the putative sequence motif — Ser (Hyp)4 — within the N-terminal end of the protein. Treatment of extensin with trifluoroacetic acid demonstrated that arabinose was the principal carbohydrate. The amino-acid composition of the purified extensin was similar to those reported in the literature. The cross-linking of extensin in vitro upon incubation with extensin peroxidase and exogenous H2O2 was characteristic of other reported extensins. Furthermore, Mono-S ion-exchange FPLC of native extensin precursor resolved it into two isoforms, A (90%) and B (10%). The amino-acid compositions of extensin A and extensin B were found to be similar to each other and both extensins were cross-linked in vitro by extensin peroxidase.