小麦叶片中1种降解Rubisco大亚基的蛋白酶鉴定

Identification of a protease related to the degradation of Rubisco large subunit in wheat leaves

[目的]探明小麦叶片衰老过程中1种降解1,5-二磷酸核酮糖羧化酶/加氧酶（ribulose 1,5-bisphosphate carboxylase/oxygenase,Rubisco）大亚基蛋白酶的生化特性。[方法]通过天然梯度聚丙烯酰胺凝胶电泳后切胶回收的方法获得Rubisco全酶,采用离子交换层析、非完全变性十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和切胶回收蛋白等方法分离目的蛋白酶,并采用含明胶的聚丙烯酰胺凝胶电泳研究其生化特性（主要是最适p H、最适温度、热稳定性、蛋白酶类型等）。[结果]切胶回收的Rubisco全酶样品中存在降解大亚基的蛋白酶。在Mono-Q离子交换层析条件下,该蛋白酶与Rubisco不易分离。利用非完全变性十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法分离出这种降解大亚基的蛋白酶;在聚丙烯酰胺凝胶中,该蛋白酶相对分子质量大于大亚基相对分子质量,位置在大亚基上方,其余部分没有检测到蛋白酶活性。该酶的最适温度为60℃;最适p H值为7.0,在p H 5.0~8.0均表现出较高的活性;该酶热稳定性一般,在50℃以上几乎完全丧失其活性;丝氨酸蛋白酶抑制剂AEBSF[4-（2-Aminoethyl）benzenesulfonyl fluoride hydrochloride]可以完全抑制蛋白酶活性,金属蛋白酶抑制剂1,10-phenanthroline可以部分抑制该蛋白酶的活性,酸性蛋白酶抑制剂pepstatin、半胱氨酸蛋白酶抑制剂E-64[transepoxysuccinyl-L-leucylamido（4-guanidino）butane]、金属蛋白酶抑制剂EDTA（ethylene diamine tetraacetic acid）和EGTA[ethylenebis（oxyethylenenitrilo）tetraacetic acid]以及丝氨酸蛋白酶抑制剂PMSF（phenylmethanesulfonyl fluoride）不抑制该蛋白酶的活性。[结论]在切胶回收的Rubisco全酶中发现了1种可以降解Rubisco大亚基的蛋白酶,其可能是1种需要金属离子的丝氨酸型蛋白酶。

[Objectives]In order to explore the mechanism of degradation of Rubisco in wheat leaves,we discovered a novel protease which could degrade Rubisco large subunit（ LSU）,and then identified the biochemical characteristics of the protease.[Methods]Rubisco holoenzyme was obtained by gel cut after natural gradient-polyacrylamide gel electrophoresis. The protease was separated by ion exchange chromatography,natural sodium dedecyl sulfate polyacrylamide gel electrophoresis（ SDS-PAGE） and gel cut. The biochemical characteristics of the protease,including the optimal temperature,optimal p H,thermal stability and the type of proteolytic enzymes,were studied by gelatin-SDS-PAGE. [Results]The LSU was degraded to many small fragments with lower molecular weight by a protease in the sample of Rubisco purified from the gel. The protease could not be separated from the sample of Rubisco holoenzymes after Mono-Q anion exchange chromatography. The protease could be separated from Rubisco after natural SDS-PAGE. The activities of different samples isolated from natural SDS-PAGE gel were detected. A protease with obvious proteolytic activity could be detected by gelatin-SDS-PAGE from the obtained sample located upon LSU. However we did not detect any protease activity in the samples of LSU,SSU,and the sample between LSU and SSU. The optimum temperature for the protease was about 60 ℃. The protease was highly active between p H 5. 0 and p H 8. 0,and the optimum p H value was around 7. 0. The protease was approximately inactive when pre-incubated at temperature higher than 50 ℃,and the protease was completely inhibited by serine protease inhibitors AEBSF. In addition,metalloprotease inhibitors of 1,10-phenanthroline could partially inhibit the activity of protease,but cysteine proteinase inhibitor E-64,metalloprotease inhibitors EDTA and EGTA,serine protease inhibitor PMSF,aspartic-type proteinase inhibitor pepstatin failed to influence the activity of protease. [Conclusions]In this paper,a novel serineprotease which could degrade the LSU was found in the sample of Rubisco holoenzymes from gel cut after natural gradient-polyacrylamide gel electrophoresis,and the protease might be a metal-dependent serine protease.