Md. Brownleader et Pm. Dey, PURIFICATION OF EXTENSIN FROM CELL-WALLS OF TOMATO (HYBRID OF LYCOPERSICON-ESCULENTUM AND LYCOPERSICON-PERUVIANUM) CELLS IN SUSPENSION-CULTURE, Planta, 191(4), 1993, pp. 457-469
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.