Ambudkar I S, Shamoo A E
Membr Biochem. 1984;5(2):119-30. doi: 10.3109/09687688409150274.
Cardiac sarcoplasmic reticulum plays a critical role in the excitation-contraction cycle and hormonal regulation of heart cells. Catecholamines exert their ionotropic action through the regulation of calcium transport into the sarcoplasmic reticulum. Cyclic 3'-5'-adenosine monophosphate (cAMP) causes the cAMP-dependent protein kinase to phosphorylate the regulatory protein phospholamban, which results in the stimulation of calcium transport. Calmodulin also phosphorylates phospholamban by a calcium-dependent mechanism. We have reported the isolation and purification of phospholamban with low deoxycholate (DOC) concentrations (5 X 10(-6) M). We have also reported the isolation and purification of Ca2+ + Mg2+-ATPase with a similar procedure. Both phospholamban and Ca2+ + Mg2+-ATPase retained their native properties associated with sarcoplasmic reticulum vesicles. Further, we have shown that the removal of phospholamban from membranes of sarcoplasmic reticulum vesicles uncouples Ca2+-uptake from ATPase without any effect on Ca2+ + Mg2+-ATPase activity or Ca2+ efflux. Phospholamban appears to be the substrate for both the Ca2+-calmodulin system and the cAMP-dependent protein kinase system. It is found that the phosphorylation of phospholamban by the Ca2+-calmodulin system is required for the normal basal level of Ca2+ transport, and that the phosphorylation of phospholamban at another site by the cAMP-dependent protein kinase system causes the stimulation of Ca2+-transport above the basal level. The functional effects of the phosphorylation of phospholamban by cAMP-dependent protein kinase system are expressed only after the phosphorylation of phospholamban with Ca2+-calmodulin system. We propose a model for the cardiac Ca2+ + Mg2+-ATPase, whereby the enzyme is normally uncoupled from Ca2+ uptake. The enzyme becomes coupled to Ca2+ transport after the first site of phospholamban is phosphorylated with the Ca2+-calmodulin system. When the second site of phospholamban is phosphorylated with cAMP-dependent protein kinase both Ca2+ transport and ATPase are stimulated and phospholamban becomes inaccessible to DOC solubilization and trypsin.
心肌肌浆网在心肌细胞的兴奋 - 收缩偶联及激素调节过程中发挥着关键作用。儿茶酚胺通过调节钙离子向肌浆网内的转运来发挥其变力作用。环磷腺苷（cAMP）促使依赖cAMP的蛋白激酶使调节蛋白受磷蛋白磷酸化，从而刺激钙离子转运。钙调蛋白也通过一种钙依赖机制使受磷蛋白磷酸化。我们报道了在低脱氧胆酸盐（DOC）浓度（5×10⁻⁶ M）下受磷蛋白的分离与纯化。我们还报道了用类似方法分离和纯化Ca²⁺ + Mg²⁺ - ATP酶。受磷蛋白和Ca²⁺ + Mg²⁺ - ATP酶都保留了与肌浆网小泡相关的天然特性。此外，我们发现从肌浆网小泡膜上去除受磷蛋白会使钙离子摄取与ATP酶解偶联，而对Ca²⁺ + Mg²⁺ - ATP酶活性或钙离子外流没有任何影响。受磷蛋白似乎是Ca²⁺ - 钙调蛋白系统和依赖cAMP的蛋白激酶系统的底物。研究发现，Ca²⁺ - 钙调蛋白系统对受磷蛋白的磷酸化是钙离子正常基础转运水平所必需的，而依赖cAMP的蛋白激酶系统在另一位点对受磷蛋白的磷酸化会使钙离子转运高于基础水平。依赖cAMP的蛋白激酶系统对受磷蛋白的磷酸化所产生的功能效应只有在受磷蛋白被Ca²⁺ - 钙调蛋白系统磷酸化之后才会表现出来。我们提出了一个关于心肌Ca²⁺ + Mg²⁺ - ATP酶的模型，即该酶通常与钙离子摄取解偶联。在用Ca²⁺ - 钙调蛋白系统使受磷蛋白的第一个位点磷酸化后，该酶与钙离子转运偶联。当用依赖cAMP的蛋白激酶使受磷蛋白的第二个位点磷酸化时，钙离子转运和ATP酶活性均受到刺激，且受磷蛋白变得不能被DOC溶解和胰蛋白酶作用。
