PURIFICATION OF EXTENSIN FROM CELL-WALLS OF TOMATO (HYBRID OF LYCOPERSICON-ESCULENTUM AND LYCOPERSICON-PERUVIANUM) CELLS IN SUSPENSION-CULTURE

Extensin, a hydroxyproline-rich glycoprotein comprising substantial am ounts of beta-L-arabinose-hydroxyproline glycosidic linkages is believ ed to be insolubilized in the cell wall during host-pathogen interacti on 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. an d L. peruvianum L. (Mill.) cells in suspension cultures and purified t o homogeneity by a rapid and simple procedure under mild and non-destr uctive experimental conditions. The molecular weight of native extensi n precursor was estimated to be greater than 240-300 kDa by Superose-1 2 gel-filtration chromatography. Extensin monomers have previously bee n designated a molecular weight of approximately 80 kDa. Our results i ndicate that salt-eluted extensin precursor is not monomeric. Agarose- gel electrophoresis, Superose-12-gel-filtration, extensin-peroxidase-c atalysed cross-linking, Mono-S ion-exchange fast protein liquid chroma tography (FPLC), and peptide-sequencing data confirmed the homogeneity of the extensin preparation. Evidence that the purified protein was e xtensin is attributed to the presence of the putative sequence motif - Ser (Hyp)4 - within the N-terminal end of the protein. Treatment of e xtensin with trifluoroacetic acid demonstrated that arabinose was the principal carbohydrate. The amino-acid composition of the purified ext ensin was similar to those reported in the literature. The cross-linki ng of extensin in vitro upon incubation with extensin peroxidase and e xogenous H2O2 was characteristic of other reported extensins. Furtherm ore, Mono-S ion-exchange FPLC of native extensin precursor resolved it into two isoforms, A (90%) and B (10%). The amino-acid compositions o f extensin A and extensin B were found to be similar to each other and both extensins were cross-linked in vitro by extensin peroxidase.