Buechler K F, Gibson D M
Arch Biochem Biophys. 1984 Sep;233(2):698-707. doi: 10.1016/0003-9861(84)90496-x.
Acetyl-CoA carboxylase was purified 300-fold from rat liver, in the absence of added citrate, by precipitation from an 18,000g supernatant in the presence of Triton X-100 at 105,000g and 20 degrees C, followed by chromatography on phosphocellulose. Acetyl-CoA carboxylase activity in this preparation was activated by preincubation with GTP (0.1-2.0 mM) and with citrate (20 mM). Colchicine (10(-6)-10(-3) M) inhibited enzyme activity and counteracted the effects of GTP and citrate. Sucrose density gradient centrifugation demonstrated that GTP and citrate preincubation promoted the formation of the polymeric, active enzyme, while colchicine engendered disassembly. Preincubation of the purified acetyl-CoA carboxylase at 4 degrees C caused inactivation and disassembly, which was countered by preincubation at 37 degrees C in the presence of GTP or citrate. These results suggest that GTP, like citrate, activates acetyl-CoA carboxylase by enhancing the conversion of the protomeric form of the enzyme to its more active, polymeric state.
在不添加柠檬酸的情况下，通过在Triton X - 100存在下从18,000g上清液于105,000g和20℃下沉淀，随后在磷酸纤维素上进行色谱分离，从大鼠肝脏中纯化出了300倍的乙酰辅酶A羧化酶。该制剂中的乙酰辅酶A羧化酶活性通过与GTP（0.1 - 2.0 mM）和柠檬酸（20 mM）预孵育而被激活。秋水仙碱（10⁻⁶ - 10⁻³ M）抑制酶活性并抵消GTP和柠檬酸的作用。蔗糖密度梯度离心表明，GTP和柠檬酸预孵育促进了聚合物形式的活性酶的形成，而秋水仙碱导致其解聚。在4℃下对纯化的乙酰辅酶A羧化酶进行预孵育会导致失活和解聚，而在37℃下于GTP或柠檬酸存在下进行预孵育可对抗这种情况。这些结果表明，GTP与柠檬酸一样，通过增强酶的单体形式向其更具活性的聚合物状态的转化来激活乙酰辅酶A羧化酶。
