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烟草Rubisco活化酶的纯化及其特性

唐如航, 贾军伟, 李立人

中科院上海植物生理研究所. 上海 200032

摘要

利用35%饱和硫酸铵分部、DEAE Sephacel和FPLC Mono Q柱层析等步骤从烟草叶片中纯化了Rubisco活化酶,并制备了其专一性抗体。此法不仅快速,而且比活力高。以往认为菠菜和拟南芥Rubisco活化酶由两种亚基组成。通过快速制备的粗提液分析,发现烟草Rubisco活化酶由一种42 kD的亚基组成。即使在有多种蛋白酶抑制剂存在的情况下, 此亚基仍很易降解为39 kD的亚基。ATP不仅对酶的活性所必需,而且也有利于维持酶的稳定性。该酶的热稳定性远比Rubisco差。

关键词： Rubisco; Rubisco活化酶; 烟草; 纯化; 特性

Purification and characterization of Rubisco activase from tobacco

Tang Ruhang, Jia Junwei, Li Liren

Shanghai Institute of Plant Physiology,The Chinese Academy of Sciences. Shanghai 200032

Abstract

Ribulose 1,5-bisphosphate carboxylase/oxygenase activase was purified from tobacco (Nicotiana tabacum) leaves by 35% ammonium sulfate fractionation, DEAE Sephacel and ion exchange fast protein liquid chromatography. A 45 fold purification with 67% recovery of activity was attained. The specific activity of purified enzyme was about 3.74 U/mg. Western blot analysis of fast prepared crude　extract showed that the tobacco Rubisco activase is composed of only　one kind of 42 kD subunit. The 39 kD polypeptide is appeared during the purification and storage of Rubisco activase.It is suggested that the 39 kD polypeptide is probably a product of breakdown of the 42 kD polypeptide. This result is different from the previous reports that Rubisco activase from spinach or arabidopsis consists of two subunits . The addition of ATP during purification was necessary to maintain Rubisco activase activity. Maximal activity of the enzyme was obtained with ATP 0.30 mmol/L in the presence of an ATP-regenerating system. ATP also enhanced the　stability of the enzyme during storage.It was found that tobacco Rubisco activase was much more labile to heat than tobacco Rubisco.

Key words: Rubiseo;Rubisco activase;Tobacco;Purification;Characterization

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引用本文：

唐如航, 贾军伟, 李立人. 烟草Rubisco活化酶的纯化及其特性[J]. 生理学报 1997; 49 (1): .

Tang Ruhang, Jia Junwei, Li Liren. Purification and characterization of Rubisco activase from tobacco. Acta Physiol Sin 1997; 49 (1): (in Chinese with English abstract).