title: Inhibition of polygalacturonase from Verticillium dahliae by a polygalacturonase inhibiting protein from cotton.
abstract: An extracellular endo-polygalacturonase (PGase) [E.C. 3.2.1.15] was isolated from 18-day-old culture filtrates of Verticillium dahliae and partially purified using gel permeation chromatography. The band responsible for PGase activity was electrophoretically characterized as having a molecular mass of approximately 29 500 and an isoelectric point of 5.4. Kinetic studies indicate a Km of 3.3 mg ml(-1) and Vmax of 0.85 micromol reducing units min(-1) ml(-1) with polygalacturonic acid as substrate. Polygalacturonase inhibitor protein (PGIP) in cotton seedlings was induced by 5 mM salicylic acid and immunochemical analysis indicated high levels in the hypocotyl tissues. PGIP was purified from roots and stems using affinity chromatography with endo-PGase from Aspergillus niger as an immobilised ligand. The purified PGIP contained monomeric and dimeric molecules with molecular masses of 34 and 66 kDa respectively. Purified cotton PGIP inhibited endo-polygalacturonase from A. niger in a non-competitive or mixed manner with an inhibition constant. K(I) of 15 nM. The isolated V. dahliae PGase was, however, inhibited in a positive cooperative manner, indicative of allosteric interactions between the enzyme and the inhibitor protein. In addition to reducing the reaction rate, decreased substrate affinity may contribute to the accumulation of elicitor-active oligouronides.
authors.affiliation: Department of Chemistry and Biochemistry, Rand Afrikaans University, Auckland Park, South Africa.
authors.forename: J T
authors.initials: JT
authors.lastname: James
authors.forename: I A
authors.initials: IA
authors.lastname: Dubery
mesh_terms: D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D003368:Gossypium / Q000737:chemistry*; D006999:Hypocreales / Q000201:enzymology*; D007700:Kinetics; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D011096:Polygalacturonase / Q000037:antagonists & inhibitors*
chemical_list: C081479:PGIP protein, plant; D010940:Plant Proteins; D011096:Polygalacturonase