Ramnanan Christopher J, Storey Kenneth B
Institute of Biochemistry, College of Natural Sciences, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6.
J Exp Biol. 2006 Feb;209(Pt 4):677-88. doi: 10.1242/jeb.02052.
Entry into the hypometabolic state of estivation requires a coordinated suppression of the rate of cellular ATP turnover, including both ATP-generating and ATP-consuming reactions. As one of the largest consumers of cellular ATP, the plasma membrane Na+/K+-ATPase is a potentially key target for regulation during estivation. Na+/K+-ATPase was investigated in foot muscle and hepatopancreas of the land snail Otala lactea, comparing active and estivating states. In both tissues enzyme properties changed significantly during estivation: maximal activity was reduced by about one-third, affinity for Mg.ATP was reduced (Km was 40% higher), and activation energy (derived from Arrhenius plots) was increased by approximately 45%. Foot muscle Na+/K+-ATPase from estivated snails also showed an 80% increase in Km Na+ and a 60% increase in Ka Mg2+ as compared with active snails, whereas hepatopancreas Na+/K+-ATPase showed a 70% increase in I50 K+ during estivation. Western blotting with antibodies recognizing the alpha subunit of Na+/K+-ATPase showed no change in the amount of enzyme protein during estivation. Instead, the estivation-responsive change in Na+/K+-ATPase activity was linked to posttranslational modification. In vitro incubations manipulating endogenous kinase and phosphatase activities indicated that Na+/K+-ATPase from estivating snails was a high phosphate, low activity form, whereas dephosphorylation returned the enzyme to a high activity state characteristic of active snails. Treatment with protein kinases A, C or G could all mediate changes in enzyme properties in vitro that mimicked the effect of estivation, whereas treatments with protein phosphatase 1 or 2A had the opposite effect. Reversible phosphorylation control of Na+/K+-ATPase can provide the means of coordinating ATP use by this ion pump with the rates of ATP generation by catabolic pathways in estivating snails.
进入夏眠的低代谢状态需要对细胞ATP周转速率进行协同抑制,这包括ATP生成反应和ATP消耗反应。作为细胞ATP的最大消耗者之一,质膜Na+/K+-ATP酶是夏眠期间调控的一个潜在关键靶点。对陆地蜗牛奥氏乳螺的足部肌肉和肝胰腺中的Na+/K+-ATP酶进行了研究,比较了活跃状态和夏眠状态。在这两种组织中,酶的性质在夏眠期间都发生了显著变化:最大活性降低了约三分之一,对Mg.ATP的亲和力降低(Km高40%),活化能(由阿伦尼乌斯曲线得出)增加了约45%。与活跃蜗牛相比,夏眠蜗牛足部肌肉的Na+/K+-ATP酶的Km Na+增加了80%,Ka Mg2+增加了60%,而肝胰腺的Na+/K+-ATP酶在夏眠期间I50 K+增加了70%。用识别Na+/K+-ATP酶α亚基的抗体进行蛋白质印迹分析表明,夏眠期间酶蛋白的量没有变化。相反,Na+/K+-ATP酶活性的夏眠响应变化与翻译后修饰有关。操纵内源性激酶和磷酸酶活性的体外孵育表明,夏眠蜗牛的Na+/K+-ATP酶是一种高磷酸化、低活性形式,而去磷酸化使该酶恢复到活跃蜗牛特有的高活性状态。用蛋白激酶A、C或G处理均可在体外介导酶性质的变化,模拟夏眠的效果,而用蛋白磷酸酶1或2A处理则产生相反的效果。Na+/K+-ATP酶的可逆磷酸化控制可以提供一种手段,使这种离子泵的ATP使用与夏眠蜗牛分解代谢途径的ATP生成速率相协调。