Bredeston Luis M, Rega Alcides F
Instituto de Química y Fisicoquímica Biológicas (Universidad de Buenos Aires-Consejo Nacional de Investigaciones Científicas y Técnicas), Facultad de Farmacia y Bioquímica, Junín 956, 1113 Buenos Aires, Argentina.
Biochem J. 2002 Jan 15;361(Pt 2):355-61. doi: 10.1042/0264-6021:3610355.
Pre-steady-state phosphorylation and dephosphorylation of purified and phospholipid-depleted plasma-membrane Ca(2+)-ATPase (PMCA) solubilized in the detergent polyoxyethylene 10 lauryl ether were studied at 25 degrees C. The time course of phosphorylation with ATP of the enzyme associated with Ca(2+), probably the true phosphorylation reaction, showed a fast phase (k(app) near 400 s(-1)) followed by a slow phase (k(app)=23 s(-1)). With asolectin or acidic phosphatidylinositol, the concentration of phosphoenzyme (EP) increased at as high a rate as before, passed through a maximum at 4 ms and stabilized at a steady level that was approx. half that without lipids. Calmodulin (CaM) did not change the rate of the fast phase, accelerated the slow phase (k(app)=93 s(-1)) and increased [EP] with small changes in the shape of the time course. Dephosphorylation was slow (k(app)=30 s(-1)) and insensitive to CaM. Asolectin accelerated dephosphorylation, which followed biexponential kinetics with fast (k(app)=220 s(-1)) and slow (k(app)=20 s(-1)) components. CaM stimulated the fast component by nearly 50%. The results show that the behaviour of the PMCA is complex, and suggest that acidic phospholipids and CaM activate PMCA through different mechanisms. Acceleration of dephosphorylation seems relevant during activation of the PMCA by acidic phospholipids.
在25℃下，研究了溶解于去污剂聚氧乙烯10月桂基醚中的纯化且去除磷脂的质膜Ca(2+) -ATP酶（PMCA）的预稳态磷酸化和去磷酸化过程。与Ca(2+)结合的酶的ATP磷酸化时间进程，可能是真正的磷酸化反应，显示出一个快速阶段（表观一级速率常数k(app)接近400 s(-1)），随后是一个缓慢阶段（k(app)=23 s(-1)）。添加大豆卵磷脂或酸性磷脂酰肌醇后，磷酸化酶（EP）的浓度以与之前相同的高速率增加，在4毫秒时达到最大值，然后稳定在一个约为无脂质时一半的稳定水平。钙调蛋白（CaM）没有改变快速阶段的速率，加速了缓慢阶段（k(app)=93 s(-1)），并增加了[EP]，同时时间进程的形状有小的变化。去磷酸化过程缓慢（k(app)=30 s(-1)），且对CaM不敏感。大豆卵磷脂加速了去磷酸化，该过程遵循双指数动力学，具有快速（k(app)=220 s(-1)）和缓慢（k(app)=20 s(-1)）成分。CaM刺激了快速成分近50%。结果表明，PMCA的行为是复杂的，提示酸性磷脂和CaM通过不同机制激活PMCA。酸性磷脂激活PMCA过程中，去磷酸化的加速似乎具有重要意义。
