Homogeneous endo-polygalacturonase (PG) was covalently bound to cyanogen-bromide-activated Sepharose, and the resulting PG-Sepharose conjugate was utilized to purify, by affinity chromatography, a protein from Phaseolus vulgaris hypocotyls that binds to and inhibits PG. Isoelectric focusing of the purified PG-inhibiting protein (PGIP) showed a major protein band that coincided with PG-inhibiting activity. PGIP formed a complex with PG at pH 5.0 and at low salt concentrations. The complex dissociated in 0.5 M Na-acetate and pH values lower than 4.5 or higher than 6.0. Formation of the PG-PGIP complex resulted in complete inhibition of PG activity. PG activity was restored upon dissociation of the complex. The protein exhibited inhibitory activity toward PGs from Colktotrichum lindemuthianum, Fusarium moniliforme and Aspergillus niger. The possible role of PGIP in regulating the activity of fungal PG's and their ability to elicit plant defense reactions are discussed.
Endo-polygalacturonase (PG) was purified from a commercial preparation of Aspergillus niger pectinase by means of carboxymethylcellulose chromatography, preparative isoelectric focusing, and gel permeation through Sephadex G-50. The enzyme was electrophoretically homogeneous and consisted of a single polypeptide chain with a molecular weight of 33,500. The enzyme exhibited a specific activity significantly higher than those of purified polygalacturonases from phytopathogenic fungi.Galacturonate oligomers with a degree of polymerization higher than four appeared quickly as products of the enzymic hydrolysis of Napolygalacturonate. The oligomers were later degraded to di-and monogalacturonate. The homogeneous enzyme and growing mycelium of Aspergillus niger separately elicited a necrotic response in cowpea (Vigna unguiculata Walp.) pods. Heat-inactivated PG and PG inactivated with specific antibodies did not elicit necrosis, suggesting that the catalytic activity of the enzyme is necessary for its function as an elicitor. The PG-released oligosaccharides from Vigna cell wall and the galacturonides with a degree of polymerization greater than four separately elicited necrosis, whereas di-and monogalacturonate did not.
The activity of extracellular polygalacturonase (PG) was strongly induced in Fusarium moniliforme by growing the fungus in a minimal medium containing pectin as sole carbon source. PG was the major protein component of the extracellular fluid whereas no detectable PG activity was found in culture filtrates of the fungus grown in a medium containing glucose as carbon source. F. moniliforme PG was purified to homogeneity by a procedure involving ammonium sulphate precipitation, carboxymethyl cellulose chromatography and preparative isoelectric focusing. The purified protein showed one protein band in non-denaturating PAGE and two major bands (molecular mass 41-5 and 45.0 kDa) plus two minor bands (38.0 and 48.5 kDa) in SDS-PAGE. The bands consisted of glycosylated polypeptide chains. Poly(A)-containing RNA, purified from total RNA extracted from F . moniliforme grown in PG-inducing conditions and translated in vitro in a reticulocyte cell-free translation system, produced two polypeptide chains (47 and 51 kDa) which were not present in the translation products of the non-induced poly(A)-containing RNA. The two polypeptide chains were immunoprecipitated with an IgG against F. moniliforme homogeneous PG.
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