Abstract
We found three types of cysteine protease from spinach leaves, which hydrolyze artificial fluorescent substrate N-succinyl-Leu-Tyr-aminomethylcoumarin (Suc-LY-AMC). They had a maximal activity at pH 5 and we named them SoCP (Spinacia oleracea Cysteine Protease) 1-3. The total activity of these enzymes increased during senescence. Conversion of SoCP2 to SoCP3 was observed and it resulted in a significant change in the molecular weight from monomer to trimer. This change also occurred in vitro, indicating that this conversion occurs not only by senescence, but also by other factors. Highly purified SoCP was a complex consisting of 41-k protein and 14-k protein. By amino acid sequences and cDNA cloning, we revealed that 41-k protein was cysteine protease containing granulin domain and 14-k protein was cystatin, an inhibitor of cysteine protease. In this study, we report on the purification and characterization of SoCP and cystatin, and also their activation and the conversion.
