Kranias E G, Steenaart N A, Di Salvo J
Department of Pharmacology, University of Cincinnati College of Medicine, Ohio 45267-0575.
J Biol Chem. 1988 Oct 25;263(30):15681-7.
A protein phosphatase which dephosphorylates phospholamban was purified from canine cardiac cytosol. Purification involved sequential chromatography on DEAE-Sephacel, polylysine-agarose, heparin-agarose, Mono Q HR 10/10, and Superose 6. The enzyme was composed of three subunits with Mr = 63,000, 55,000, and 38,000, and it could dephosphorylate the sites on phospholamban phosphorylated by either cAMP-dependent or calcium-calmodulin-dependent protein kinase. Phospholamban phosphatase activity was enhanced 12-, 9-, and 3-fold by the divalent cations Mg2+, Mn2+, and Ca2+, respectively. The phosphatase was inhibited by PPi, ATP, NaF, and Pi and the degree of inhibition was different with each compound. The substrate specificity of the purified phosphatase for cardiac phosphoproteins was determined using troponin I, phospholamban, and highly enriched sarcolemmal and sarcoplasmic reticulum preparations, phosphorylated by the cAMP-dependent protein kinase. The phosphatase exhibited the highest activity with phospholamban as substrate. Thus, dephosphorylation of phospholamban by this phosphatase may participate in regulation of sarcoplasmic reticulum function in cardiac muscle.
从犬心肌细胞溶质中纯化出一种使受磷蛋白去磷酸化的蛋白磷酸酶。纯化过程包括依次在DEAE-葡聚糖凝胶、聚赖氨酸琼脂糖、肝素琼脂糖、Mono Q HR 10/10和Superose 6上进行层析。该酶由三个亚基组成，其分子量分别为63,000、55,000和38,000，并且它能够使受磷蛋白上被环磷酸腺苷（cAMP）依赖性蛋白激酶或钙调蛋白依赖性蛋白激酶磷酸化的位点去磷酸化。受磷蛋白磷酸酶活性分别被二价阳离子Mg2+、Mn2+和Ca2+增强了12倍、9倍和3倍。该磷酸酶被焦磷酸（PPi）、三磷酸腺苷（ATP）、氟化钠（NaF）和无机磷酸盐（Pi）抑制，并且每种化合物的抑制程度不同。使用肌钙蛋白I、受磷蛋白以及经cAMP依赖性蛋白激酶磷酸化的高度富集的肌膜和肌浆网制剂，测定纯化的磷酸酶对心脏磷蛋白的底物特异性。该磷酸酶以受磷蛋白作为底物时表现出最高活性。因此，这种磷酸酶使受磷蛋白去磷酸化可能参与心肌中肌浆网功能的调节。
