Inhibition of animal and pathogenic fungal proteases by phloem exudate from pumpkin fruits (cucurbitaceae)

Abstract

Abstract Healthy pumpkin fruits exude a gel‐forming liquid when the skin is cut. This exudate inhibits trypsin activity (EC 3.4.21.4). The inhibitor differs from p ‐protein in not being precipitated by aeration nor by heating. One inhibitory zone at approximately pH 5 was detected by isoelectric focusing. Using polyacrylamide gel electrophoresis (PAGE), one major and four minor bands of inhibitor were detected. With sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), the inhibitor moved with the tracker dye, indicating a molecular weight of less than 6000 daltons. Inhibition of trypsin activity was noncompetitive. Pumpkins of longer keeping cultivars generally had higher levels of inhibitor. The concentration of inhibitor activity in pumpkin flesh was approximately one thousandth that of phloem exudate, but exudate from the stems of pumpkin seedlings contained inhibitor at similar concentrations to fruit exudate. Protease obtained from the fungal rot regions of pumpkins was, like trypsin itself, inhibited by the exudate material. Since high levels of antitrypsin factors have been associated with disease resistance in other plant/pathogen combinations, it is suggested that this inhibition provides a protective mechanism aganst fungi causing rot of pumpkins.