烟草Rubisco活化酶的纯化及其特性

利用35％饱和硫酸铵分部、DEAE－Sephacel和FPIC－MonoQ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42kD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39kD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。

Purification and Characterization of Rubisco Activase from Tobacco

Ribulose 1, 5-bisphosphate carboxylase/oxygenase activase was purified from tobacco (Nicotiana tabacum) leaves by 35% ammonium sulfate fractionahon, DEAE-fephacel 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(Table 1). Western blot analysis of fast prepared crude extract showed that the tobacco Rubisco activase is composed of only one kind of 42 all subunit. The 39 kD polypephde is appeared during the purification and storage of Rubisco activate. It is suggested that the 39 kD polypeptide is probably a product of breakdown of the 42 all polypeptide. TIs result is different from the previous reports that Rubisco achvase from spinach or arabiolopsis consists of two subunitS (Fig. 2).The addition of Aam during purification was necessary to maintain Rubisco achvase activity (Fig. 6). Mixirnal activity of the enzyme was obtained with ATh 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 achvase was much more labile to heat than tobacco Rubiseo (Fig. 7 ).