Research on the prolyl endopeptidase of thylakoid membranes of chloroplasts.

叶绿体类囊体膜脯氨酰内肽酶的研究。

• 批准号: 62540515
• 负责人: KUWABARA Tomohiko
• 金额: $ 1.15万
• 依托单位: Toho University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1987
• 资助国家: 日本
• 起止时间: 1987 至 1988
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-62540515/
• 关键词: Protease; Thylakoid; Membrane; Photosystem II; Extrinsic protein; ホウレンソウ; 光化学系II; チラコイド膜; 光化学系II粒子; 葉緑体

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.

本研究证实：（1）菠菜PSII膜上存在多种蛋白酶。(2)一种蛋白酶切割PSII水氧化复合物的23-KDa蛋白中的Phe-Gly键，并且对SH-试剂敏感。(3)另一种蛋白酶可能是脯氨酰内肽酶（E.C.3.4.21.26），因为PSII水氧化复合物的18-KDa蛋白质中的pro-Ile和Pro Leu键被切割。由于脯氨酰内肽酶的抑制剂氟磷酸二异丙酯和N-苄氧羰基-L-硫代脯氨酰-噻唑烷对脯氨酰内肽酶的抑制作用不明显，因此该蛋白酶可能是一种新型的外源蛋白酶。使用18-KDa蛋白作为底物最好地测定蛋白酶活性。在凝胶过滤层析后，该蛋白酶在156 KDa和39 KDa处显示出两个峰。经SDS-PAGE分析，两个峰均为39 KDa的多肽。其中一个峰的再层析产生了两个峰。这些结果表明，该蛋白酶与39-KDa亚基形成四聚体。

项目成果

Tomohiko Kuwabara: "Purification of an extrinsic protease from photosystem II membranes of spinach chloroplasts."

Tomohiko Kuwabara：“从菠菜叶绿体光系统 II 膜中纯化外源蛋白酶。”

Tomohiko Kuwabara: "Association of extrinsic proteases with the photosystem II membranes prepared with Triton X-100 from spinach chloroplasts."

Tomohiko Kuwabara：“外源蛋白酶与用 Triton X-100 从菠菜叶绿体制备的光系统 II 膜的关联。”