Poster: Enzymology

Abs # 248: Purification and Characterization of a Senescence Activated Acid Protease from Spinach Leaves

Presenter:	Satoh, Masashi , r0235009@ipc.shizuoka.ac.jp
Authors	Satoh, Masashi  (A)   Amano, Toyoki  (A)   Shioi, Yuzo  (A)
Affiliations:	(A): Department of Biology and Geoscience, Faculty of Science, Shizuoka University

Leaf senescence is the final stage of leaf development. During this stage, various proteases are involved, and it is known that cysteine protease plays an important role in proteolysis during senescence. In this study, we partially purified the acid protease that was activated by senescence from spinach leaves. On the basis of its proteolytic activity towards fluorogenic peptide, Suc-Leu-Tyr-AMC (SLT-AMC), the purification was carried out using anion exchange, hydrophobic, and gel-filtration chromatography. Molecular weight of the protease was estimated to be 26,000 by SDS-PAGE. Proteolytic activity of the protease was markedly activated by artificial senescence treatment. The protease activity was also increased by SDS (optimum conc. 0.2%), but the process of the purification, this activation decreased. The protease had a pH optimum around 5.0, and proteolytic activity was inhibited by N-ethylmaleimide (NEM), suggesting that the enzyme is one of the cysteine proteases. From these findings, a likely conclusion is that a novel type of senescence-induced protease is present in spinach. In order to investigate the properties of the protease, further purification and kinetical analyses of the protease are now in progress.