Ferrington Deborah A, Yao Qing, Squier Thomas C, Bigelow Diana J
Department of Biochemistry, University of Kansas, Lawrence, Kansas 66045, USA.
Biochemistry. 2002 Nov 5;41(44):13289-96. doi: 10.1021/bi026407t.
Alterations in expression levels of phospholamban (PLB) relative to the sarcoplasmic reticulum (SR) Ca-ATPase have been suggested to underlie defects of calcium regulation in the failing heart and other cardiac pathologies. To understand how variation in PLB expression relative to that of the Ca-ATPase can modulate calcium transport, we have investigated the inhibition of the Ca-ATPase by PLB in native SR membranes from slow-twitch skeletal and cardiac muscle and in reconstituted proteoliposomes. Quantitative immunoblotting in combination with affinity-purified protein standards was used to measure protein concentrations of PLB and of the Ca-ATPase. Functional inhibition of the Ca-ATPase was determined from both the calcium concentrations for half-maximal activation (Ca(1/2)) and the shift in the calcium concentrations following release of PLB inhibition (i.e., (Delta)Ca(1/2)) by incubation with monoclonal antibodies against PLB, which are equivalent to phosphorylation of PLB by cAMP-dependent protein kinase. We report that equivalent levels of PLB inhibition and antibody-induced activation ((Delta)Ca(1/2) = 0.25 +/- 0.02 microM) are observed in SR membranes from slow-twitch skeletal and cardiac muscle, where molar stoichiometries of PLB expressed per Ca-ATPase vary, respectively, from 0.9 +/- 0.1 to 4.1 +/- 0.8. Similar levels of inhibition to those observed in isolated SR vesicles were observed using reconstituted proteoliposomes following co-reconstitution of affinity-purified Ca-ATPase with PLB. These results indicate that total expression levels of one PLB per Ca-ATPase result in full inhibition of the Ca-ATPase and, based on the measured K(D) (140 +/- 30 microM), suggests one PLB complexed with two Ca-ATPase molecules is sufficient for full inhibition of activity. Therefore, the excess PLB expressed in the heart over that required for inhibition suggests a capability for graded responses of the Ca-ATPase activity to endogenous kinases and phosphatases that modulate the level of phosphorylation necessary to relieve inhibition of the Ca-ATPase by PLB.
相对于肌浆网（SR）钙 - ATP酶而言，受磷蛋白（PLB）表达水平的改变被认为是导致衰竭心脏及其他心脏病变中钙调节缺陷的原因。为了了解相对于钙 - ATP酶而言，PLB表达的变化是如何调节钙转运的，我们研究了慢肌骨骼肌和心肌天然SR膜以及重组蛋白脂质体中PLB对钙 - ATP酶的抑制作用。结合亲和纯化的蛋白质标准品进行定量免疫印迹，以测量PLB和钙 - ATP酶的蛋白质浓度。通过与抗PLB单克隆抗体孵育，从半数最大激活的钙浓度（Ca(1/2)）以及PLB抑制解除后钙浓度的变化（即(Delta)Ca(1/2)）来确定钙 - ATP酶的功能抑制，这等同于cAMP依赖性蛋白激酶对PLB的磷酸化作用。我们报道，在慢肌骨骼肌和心肌的SR膜中观察到等量的PLB抑制和抗体诱导激活（(Delta)Ca(1/2) = 0.25 +/- 0.02 microM），其中每个钙 - ATP酶所表达的PLB摩尔化学计量分别在0.9 +/- 0.1至4.1 +/- 0.8之间变化。在将亲和纯化的钙 - ATP酶与PLB共同重组后，使用重组蛋白脂质体观察到与分离的SR囊泡中相似水平的抑制作用。这些结果表明，每个钙 - ATP酶一个PLB的总表达水平会导致钙 - ATP酶完全抑制，并且基于测得的K(D)（140 +/- 30 microM），表明一个与两个钙 - ATP酶分子复合的PLB足以完全抑制活性。因此，心脏中表达的PLB超过抑制所需水平，这表明钙 - ATP酶活性对内源激酶和磷酸酶具有分级反应的能力，这些激酶和磷酸酶可调节缓解PLB对钙 - ATP酶抑制所需的磷酸化水平。
