Purification And Characterization Of The Proteases Associated PSII Particles Of Spinach

Protein degradation is one of the major regulatory processes that allow the adaptation, repair, or removal of mutated or damaged thylakoid proteins during environmental or developmental changes. Intracellular proteases play an essential role in the turnover of intracellular proteins. They are involved both in the production of mature proteins, acting as processing enzymes, and in the degradation of damaged or non-functional proteins [1"8l In plant plastids, proteases function in protein degradation seem to act to adjust the stoichiometry of subunits in supramolecular complexes, as well as to regulate the stoichiometry between different supramolecular complexes in response to environmental changes and process the nuclear-encoded preproteins in the stromal. Large-scale hydrolysis of proteins occurs early in leaf senescence and is likely to dependent on the integrated action of groups of enzymes. The substrate for proteases in senescing leaves includes mainly enzymes and membrane proteins that function in photosynthesis. Nowadays, the reported proteases in thykaloids include the energy-enzyme and non-energy-enzyme. They are almost all involved in the processing and degradation of proteins in lumen or membrane of thykaloid [12-18].In our lab, some proteases associated with photosystem II of spinach have been discovered in years. These proteases were different with the ones reported in past and it suggests that there may be more proteases associated in PSII of chloroplasts of spinach. Such as, one of them could be resistant to 1 mol/L NaCl and be almostinhibited by 1 mmol/L DTT or mercaptoethanol. It could cleave the 18 kD peptide of PSII and produce smaller peptides [5]. In order to detect the proteases in the NaCl extract of PSII particles, the SDS-gelatin-PAGE was applied in our experience, to identify and characterize proteases in the 1 mol/L NaCl extract of spinach PSII particles. Six proteases with molecular mass of 34, 37, 50, 54, 58 kD were detected from NaCl extract of PSII particles. Among these actively proteases, a new component of the chloroplast proteolytic machinery was identified as a novel protease.. The molecular mass of the protease was identified as 58-kD by the activity gel assay. The purified enzyme was a monomeric protein whose optimum pH was 8.0. Inhibitor study demonstrated that it was a metalloprotease and contained sulfhydryl groups essential for catalytic activity. Its activity was inhibited by divalent cations such as Zn2+, Mn2+, and the Kj values of Zn2+, Mn2+were ImM. Inhibition by Zn2+ and Mn2+ additive, and almost 100% of the activity was inhibited under their physiologically significant concentrations. These results suggest that the protease is possibly regulated by divalent cations in vivo. The metalloprotease may be involved in the turnover of the lumen and membrane proteins in thylakoids.