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Study of a tomato protein which inhibits endo-polygalacturonase from Verticillium albo-atrum

Li-Kuang Huang, University of Massachusetts Amherst

Abstract

A polygalacturonase-inhibiting protein (PGIP) was purified from tomato fruit and stem tissue and was subsequently characterized. PGIP was found in stem, leaf and fruit tissue of tomato but not in root tissue. The PGIP was purified by a combination of ion-exchange, gel filtration and hydrophobic interaction chromatography. The purified tomato fruit PGIP appeared on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a broad band with a molecular weight range between 37 kDa and 43 kDa. Its molecular weight as estimated by gel filtration was 43 kDa. It stained positive for the presence of carbohydrate and had a pI of greater than 8.6. The PGIP was active against Verticillium albo-atrum endo-polygalacturonase (endo-PG) and, to a lesser extent, Aspergillus niger endo-PG. It did not inhibit crude endo-PG preparations from several other fungi or tomato endo-PG. The PGIP was stable in the pH range 3-13 and its activity was essentially constant in the pH range 3.8-6.9. It was stable at temperatures up to 50$\sp\circ$C. Preincubation of PGIP with endo-PG before adding substrate solution enhanced PGIP activity. The endo-PG from V. albo-atrum was purified to electrophoretic homogeneity on SDS-PAGE. The enzyme was capable of hydrolyzing several pectic substrates, including trigalacturonate, polygalacturonate, pectin with 31%, 56%, 70% and 93% esterification, and pectin in cell walls from tomato fruit. Non-denaturing PAGE and affinity chromatography were used to study the interaction of the enzyme and tomato PGIP. In both systems, it was demonstrated that the substrate, i.e., polygalacturonate, was required for the formation of an enzyme-inhibitor complex. In the presence of tomato PGIP, endo-PG acted on polygalacturonate and tomato cell walls in a similar way but at a much slower pace. Consequently, the presence of oligogalacturonides which can act as phytoalexin elicitors was extended.

Subject Area

Plant pathology|Biochemistry

Recommended Citation

Huang, Li-Kuang, "Study of a tomato protein which inhibits endo-polygalacturonase from Verticillium albo-atrum" (1996). Doctoral Dissertations Available from Proquest. AAI9709605.
https://scholarworks.umass.edu/dissertations/AAI9709605

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