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

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Abstract

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.

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Abbreviations

CM-cellulose:

carboxymethyl-cellulose

FPLC:

fast protein liquid chromatography

HF:

hydrogen fluoride

HRGP:

hydroxyproline-rich glycoprotein

Hyp:

hydroxyproline

Vc :

retention volume

TCA:

trichloroacetic acid

TFA:

tri-fluoroacetic acid

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Author information

Correspondence to Prakash M. Dey.

Additional information

This work was supported by a A.F.R.C. postdoctoral assistantship to Michael D. Brownleader. We thank Dr. Anthony K. Allen (Department of Biochemistry, Charing Cross and Westminster Hospital, London, UK) for performing the amino-acid analysis and Mrs. Margaret Pickering (Department of Biochemistry, Royal Holloway) for performing the peptide-sequence analysis of extensin. We also express our gratitide to Dr. A. Mort (Oklahoma State University) for performing the HF-deglycosylation of extensin.

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Brownleader, M.D., Dey, P.M. Purification of extensin from cell walls of tomato (hybrid of Lycopersicon esculentum and L. peruvianum) cells in suspension culture. Planta 191, 457–469 (1993). https://doi.org/10.1007/BF00195747

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Key words

  • Cell wall
  • Extensin
  • Extensin peroxidase
  • Hydroxyproline-rich glycoprotein
  • Lycopersicon (cell culture)