Research on the prolyl endopeptidase of thylakoid membranes of chloroplasts.

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This work calrified followings: (1) More than one proteases are extrinsically associated with spinach PSII membranes. (2) One protease cleaves a Phe-Gly bond in the 23-KDa protein of PSII water-oxidation complex, and is sensitive to SH-reagents. (3) Another protease is likely to be a prolyl endopeptidase (E.C.3.4.21.26), since a pro-Ile and a Pro Leu bonds in the 18-KDa protein of PSII water-oxidation complex were cleaved. The protease seems to be a new type, since inhibitors specific to prolyl endopeptidases, diisopropyl fluorophosphate and N-benzyloxycarbony1-L- thioproly1-thiazolidine, were ineffective.An extrinsic protease was purified from spinach PSII membranes by hydrophobic, anion-exchange and gel filtration chromatography. Protease activity was best assayed using the 18-KDa protein as the substrate. Upon gel filtration chromatography, the protease exhibited two peaks at 156 KDa and 39 KDa. Both peaks showed a polypeptide of 39 KDa on SDS-PAGE. Re-chromatography of either peak gave birth to the both of the two peaks. These results suggest that the protease forms a tetramer with the 39-KDa subunit.