Purification and characterization of polygalacturonase inhibiting proteins (PGIP) from two varieties of Asian pear
Jianbao Tian, Maria Pastore. Carl Greve, Ariel Vicente, Roberto Gregori, and John M. Labavitch
Abstracts of 27th International Horticultural Congress & Exhibition (IHC 2006), August 13-19, 2006, COEX (Convention & Exhibition), Seoul, Korea. 494 pages.
2006
บทคัดย่อ
Purification and characterization of polygalacturonase inhibiting proteins (PGIP) from two varieties of Asian pear
PGIPs are plant proteins that inhibit fungal polygalacturonases (poly (1, 4-D galacturonide) glycanohydrolases ; EC 3.2.1.15).PGIPs are thought to contribute to plant defense responses against pathogens.Plant material, Asian pears cvs.Shinli and Shinko were frozen in liquid N2 and used immediately or stored at –20°C until use.PG sources from Asper-gillus niger commercial pectinase (Sigma Aldrich, USA), Botrytis cinerea and Colletotrichum acutatum were used as the sources of PGs.PGIP extraction 30 gr of Shinli and Shinko pear fruits were processed.Ex-tracts were dialyzed overnight against buffer, and then used toassay PGIP activity.Inhibition of endo-PG activity from A. niger commercial pectinase (Sigma Aldrich, USA), C. acutatum and B. cinerea was assayed, using a gel diffusion assay according to Taylor and Secor, (1988).For PGIP purification fruit were homogenized in an equal volume of extraction buffer, and applied to a column of Con A-Sepharose 48.Chromatography was performed at 0.5 ml/min.Protein bound to the column was eluted using 750 mM methyl a D-manno pyranoside in Con A buffer.The eluent was dialyzed against 50 mM sodium acetate, pH 4.5 (buffer A), and then concentrated by ultrafiltraton using a pressure cell fitted with a PM-10 membrane (Amicon, Danvers, MA).Asian pear cultivars Shinli and Shinko showed different PGIP inhibition against different PGs from distinct organisms; PGIP inhibition activity was strictly pH dependent against A.niger. atpH 5.75; it was not significantly affected by ionic strength; but susceptible to temperature, and reduced by 80-90% at 80°C for 10 minutes.It was neither susceptible to extracting time, nor to NaCI content in extracting buffer.Its activity from different tissues was completely different (fruits < spurs >flowers > leaves).It was a sugar bound protein.However, it was fractioned by SEC at its size 4.8 x 105, but it was not sensitive to IEC.