|Budget Amount *help
¥5,900,000 (Direct Cost: ¥5,900,000)
Fiscal Year 2003: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 2002: ¥3,300,000 (Direct Cost: ¥3,300,000)
Oryzacystatin (OC) contained within rice (Oryza sativa) is the protein, which inhibits cysteine protease (CP) specifically. It inhibits papain stoichiometrically and has a great thermostability, its activity still remain even if under the conditions of cooking rice, and also is stable with acid and alkali. It regulates the metabolism of seed by controlling the activity of CP, which is concerned with protein degradation in seed. OC also recognizes the CP that exists in foreigners or sources of infection like insect, bacteria and virus as an extrinsic target enzyme and inhibits their activity, so it is said to play an important role in biological defense.
This study is focused on function-structure correlation analysis of OC using biochemical method and three-dimensional structural analysis. We succeeded in conversion between dimer and monomer of OC freely by controlling the temperature and pH of solution or concentration of protein or by addition of denaturing agent. We revealed that dimer has less inhibitory activity than monomer. This result suggests that OC acts as a regulator, which regulates protease activity by its conformational change depending on various environmental changes in the stages of development of rice. Solution NMR structure of OC revealed the conformational change occurred with the regions of the first loop and the second loop, which are important for inhibition activity, when it is dimerized. This change would be the cause of the decrease of inhibition activity. In addition, we succeeded in crystallization with homodimer of OC and diffraction data was collected to 2.9 A.
OC is applicable to drug medicine and functional food. It became clear that the activity of OC could be regulated by change the solution condition in this study, so we can use this mechanism for construction of nanomachine controlled by pH, and have resistance to acid and heat.