Research on the prolyl endopeptidase of thylakoid membranes of chloroplasts.

本工作的主要结论如下：(1)菠菜PSII膜上存在多种外源蛋白水解酶。(2)有一种酶能裂解PSII水氧化复合体的23-KDa蛋白中的Phe-Gly键，并对SH试剂敏感。(3)由于PSII水氧化复合体18-KDa蛋白中的前Ile键和Pro Leu键被切割，因此另一种酶可能是Pro内肽酶(E.C.3.4.21.26)。通过疏水层析、阴离子交换层析和凝胶过滤，从菠菜PSII膜中分离纯化出一种外源蛋白水解酶。以18-KDa蛋白为底物测定酶活力最好。经凝胶过滤层析，该酶在156 KDa和39 KDa处有两个峰。两个峰在SDS-PAGE上均显示39 KDa的多肽。两个峰的再层析都产生了两个峰。这些结果表明，该酶与39-KDa亚基形成一个四聚体。

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.