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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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Citing This Article：

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).