番茄果实中焦磷酸:D-果糖-6-磷酸 1-磷酸转移酶的两种酶型的分离纯化及其特性

Purification of Two Forms of PFP from Tomato Fruit and Their Properties

用快速蛋白液相层析仪(FPLC)Mono Q柱(HR5/5)分离纯化成熟绿番茄果实中PFP的两种分子酶型及其特性。一种酶型为Q_1,是含两个β-亚基(60kD)的二聚体,比活为5μmol min^(-1) mg^(-1);另一种为Q_2,由四个α-亚基(66kD)和四个β-亚基(60kD)组成八聚体,比活为70.5μmol/min^(-1)·mg^(-1)。Q_1的分子量是120kD,Q_2的分子量介于500kD和530kD之间。用纯化的Q_2制备的抗血清专一地与Q_2起沉淀反应。PFP酶液贮存后,其Q_1/Q_2蛋白量比值增加明显,表明部分Q_2转化为Q_1。Q_1具有催化活力表明PFP的活性中心位于β-亚基。α-亚基可能借增强PFP酶对F2,6P_2的亲和力以提高酶的比活而起调节功能,但是Q_1的活力依赖于F2,6P_2的激活,表明β-亚基处也可能存在F_2,_6P_2的调节位点。Q_2含紧密结合的F2,6P_2分子,并表现出对F2,6P2_的不敏感性,基于此种现象,有必要重新认识PFP对F2,6P_2敏感性的内在实质。

Two forms of pyrophosphate fructose-6-phosphate 1-phosphotransferase were purified from green tomato (Lycopersicon esculentum cv. T8282) fruits by chromatography on a FPLC Mono Q, column (HR5/5) and their properties studied. One form (designated Q_1) contained only two β-(60 kD) subunits, the other form (designated Q_2) was consisted of four α-(66 kD) subunits and four β-(60 kD) subunits. Native gradient PAGE revealed that the apparent relative molecular weight (Mr) value of Q_1 was 120 kD and that of Q_2 was between 500 kD and 530 kD. Antiserum to Q_2 cross-reacted with Q_2 in Ouchterlony double diffusion experiment. Q_1/Q_2 protein ratio increased markedly during storage, indicating convertion from Q_2 to Q_1.O_1 showed catalytic activity, suggesting that the activity centre of PFP rests on the β-subunits and α-subuninits may enhance the affinity of F2, 6P_2 for Q_2 and thus increase the specific activity of PFP. High activation of Q_1 by F2, 6P_2 does not rule out the possibility of some regulative sites on β-subuits; low activation of Q_2 by F2, 6P_2 was probably caused by tight binding of F2, 6P_2 to Q_2.