Purification and characterization of a thiol-protease induced during senescence of unpollinated ovaries of Pisum sativum

A senescence-specific protease has been purified from senescent unpollinated ovaries of Pisum sativum L. cv. Alaska by acidic extraction. (NH₄)₂SO₄ fractionation, ion exchange chromatography on CM-Sephadex, and affinity chromatography on ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)-Sepharose. Characterization of the purified protease indicated that it is a thiol-endoprotease (EC 3. 4. 22 class) active over a wide pH range. Purified antibodies against this protease inhibit the degradation of Rubisco in autodigested extracts of senescent ovaries, suggesting that Rubisco might be a substrate for the protease in senescent pea ovaries. The relative levels of the protease were determined by an enzyme-linked immunosorbent assay (ELISA) along the processes of ovary senescence and gibberellic acid (GA)-induced fruit development, indicating its induction at the beginning of senescence and the suppression of its synthesis by GA treatment.