Purification of extensin from cell walls of tomato (hybrid of Lycopersicon esculentum and L. peruvianum) cells in suspension culture.
Extensin, a hydroxyproline-rich glycoprotein comprising substantial amounts of beta-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.
Brownleader
M D
MD
Department of Biochemistry, University of London, Egham, Surrey, UK.
Dey
P M
PM
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002473:Cell Wall / Q000737:chemistry*; D002478:Cells, Cultured; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification*; D006031:Glycosylation; D006909:Hydroxyproline / Q000032:analysis; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011498:Protein Precursors / Q000378:metabolism; D014675:Vegetables / Q000737:chemistry*
D000596:Amino Acids; D006023:Glycoproteins; D010940:Plant Proteins; D011498:Protein Precursors; C005488:extensin protein, plant; D010544:Peroxidases; D006909:Hydroxyproline