Abstract
We previously reported that Rubisco-LSU was specifically cleaved at the peptide bond between Phe-40 and Arg-41 in lysates of wheat chloroplasts incubated in darkness. In this study, we attempted to purify and identify the protease localizing in chloroplasts which involves in the degradation of Rubisco-LSU at the same site. We first prepared the fluorescence quenching substrate having the proximal sequence of the cleavage site of the LSU to detect the protease activity. The protease was purified from 500 g of spinach leaves through ammonium sulphate precipitation, hydrophobic interaction chromatography and gel filtration. Protease activity was inhibited by EDTA and o-phenanthroline. SDS-PAGE analysis showed two polypeptides (MW 80 kDa, 103 kDa) were involved in the final fraction. Then we isolated each polypeptide, and determined their partial amino acid sequences. The amino acid sequence of 103 kDa polypeptide was identical one of the metalloproteases found in Arabidopsis thaliana.
