Laboratório de Físico-Química Biológica Aída Hassón-Voloch, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal de Rio de Janeiro, 21941-902 Rio de Janeiro, Brazil.
Int J Biochem Cell Biol. 2011 Mar;43(3):358-62. doi: 10.1016/j.biocel.2010.11.004. Epub 2010 Nov 21.
Copper-stimulated P-type ATPases are essential in the fine-tuning of intracellular copper. In the present work we characterized a copper-dependent ATPase hydrolysis in a native Golgi-enriched preparation from liver and investigated its modulation by cyclic AMP-dependent protein kinase (PKA). The very high-affinity Atp7b copper pump presented here shows a K(0.5) for free copper of 2.5×10(-17) M in bathocuproine disulfonate/copper buffer and ATP hydrolysis was inhibited 50% upon stimulation of PKA pathway, using forskolin, cAMP or cholera toxin. Incubation with PKA inhibitor (PKAi(5-24) peptide) raises Cu(I)-ATPase activity by 50%. Addition of purified PKA α-catalytic subunit increases K(0.5) for free copper (6.2×10(-17) M) without modification in the affinity for ATP in the low-affinity range of the substrate curve (∼1 mM). The Hill coefficient for free copper activation also remains unchanged if exogenous PKA is added (2.7 and 2.3 in the absence and presence of PKA, respectively). The results demonstrate that this high-affinity copper pump in its natural environment is a target of the liver PKA pathway, being regulatory phosphorylation able to influence both turnover rate and ion affinity.
铜刺激的 P 型 ATP 酶在细胞内铜的微调中是必不可少的。在本工作中，我们对来自肝脏的天然高尔基体富集制剂中的铜依赖性 ATP 水解进行了表征，并研究了其环 AMP 依赖性蛋白激酶（PKA）的调节作用。这里呈现的高亲和力 Atp7b 铜泵对游离铜的 K(0.5) 在浴铜灵/铜缓冲液中为 2.5×10(-17) M，使用福司可林、cAMP 或霍乱毒素刺激 PKA 途径时，ATP 水解被抑制 50%。用 PKA 抑制剂（PKAi(5-24) 肽）孵育可使 Cu(I)-ATPase 活性提高 50%。添加纯化的 PKA α-催化亚基会增加游离铜的 K(0.5)（6.2×10(-17) M），而不改变低亲和力底物曲线（∼1 mM）中 ATP 的亲和力。如果添加外源性 PKA，游离铜激活的 Hill 系数也保持不变（分别为 2.7 和 2.3）。结果表明，这种天然环境中的高亲和力铜泵是肝脏 PKA 途径的靶标，调节磷酸化能够影响周转率和离子亲和力。
