Comparison of the kinetic effects of phospholamban phosphorylation and anti-phospholamban monoclonal antibody on the calcium pump in purified cardiac sarcoplasmic reticulum membranes.
Comparison of the kinetic effects of phospholamban phosphorylation and anti-phospholamban monoclonal antibody on the calcium pump in purified cardiac sarcoplasmic reticulum membranes.
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受磷蛋白磷酸化和抗受磷蛋白单克隆抗体对纯化心脏肌浆网膜中钙泵的动力学影响的比较。
DOI：
10.1021/bi971109v
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Kirchberger,MA
中科院分区：
文献类型：
--
作者：
Antipenko,AY;Spielman,AI;Sassaroli,M;Kirchberger,MA
受磷蛋白（phospholamban，PLN）是心脏钙泵的蛋白调节剂，在β-肾上腺素能受体激动剂作用下，蛋白激酶A-（PKA-）催化PLN磷酸化介导心脏收缩缩短。然而，以前的研究人员一直未能成功地证明PLN磷酸化或抗PLN单克隆抗体（mAb）的作用，这被认为是模仿磷酸化的众所周知的作用对Km（Ca），微粒体钙摄取在（高）钙浓度发现在峰值收缩期细胞内。因此，我们比较了PKA的催化亚基和抗PLN mAb对蔗糖梯度纯化的心脏微粒体中钙摄取动力学的影响。在25 ° C和37 °C下，两种处理都使Vmax（Ca）增加了33 - 44%，Km（Ca）降低了11 - 31%，而Ca 2 +-ATP酶活性的变化相当。基于不同温度下E2 P分解和稳态E2 P形成的动力学测量，PLN磷酸化后E2 P分解的加速可能有助于在25 °C下而不是在37 °C下增加的Ca摄取的Vmax（Ca）。我们的数据文件几乎相同的增加inVmax（Ca）的微粒体钙摄取与PLN磷酸化或添加抗PLN单克隆抗体，因此提供了深入了解PLN的调节心肌肌浆网钙泵蛋白的动力学机制。
Protein kinase A- (PKA-) catalyzed phosphorylation of phospholamban (PLN), the protein regulator of the cardiac Ca pump, mediates abbreviation of systole in response to β-adrenergic agonists. Investigators previously, however, have been unsuccessful in demonstrating an effect of PLN phosphorylation or anti-PLN monoclonal antibody (mAb), which is considered to mimic phosphorylation's well-known effect onKm(Ca), on microsomal Ca uptake at the (high) Ca2+concentrations found intracellularly at peak systole. We therefore compared the effects of the catalytic subunit of PKA and anti-PLN mAb on the kinetics of Ca uptake in sucrose gradient-purified cardiac microsomes. Both treatments produced a 33−44% increase inVmax(Ca)at 25 and 37 °C, and an 11−31% decrease inKm(Ca)with comparable changes in Ca2+-ATPase activity. An acceleration of E2P decomposition upon PLN phosphorylation may contribute to the increasedVmax(Ca)of Ca uptake at 25 °C but not at 37 °C, based on measurement of the kinetics of E2P decomposition and steady-state E2P formation from Piat different temperatures. Our data document almost identical increases inVmax(Ca)of microsomal Ca uptake with PLN phosphorylation or addition of anti-PLN mAb and hence provide insight into the kinetic mechanism of PLN's regulation of the cardiac sarcoplasmic reticulum Ca pump protein.