Steenaart N A, Ganim J R, Di Salvo J, Kranias E G
Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Ohio 45267-0575.
Arch Biochem Biophys. 1992 Feb 14;293(1):17-24. doi: 10.1016/0003-9861(92)90359-5.
Canine cardiac sarcoplasmic reticulum vesicles contain intrinsic protein phosphatase activity, which can dephosphorylate phospholamban and regulate calcium transport. This phosphatase has been suggested to be a mixture of both type 1 and type 2 enzymes (E. G. Kranias and J. Di Salvo, 1986, J. Biol. Chem. 261, 10,029-10,032). In the present study the sarcoplasmic reticulum phosphatase activity was solubilized with n-octyl-beta-D-glucopyranoside and purified by sequential chromatography on DEAE-Sephacel, polylysine-agarose, heparin-agarose, and DEAE-Sephadex. A single peak of phosphatase activity was eluted from each column and it was coincident for both phospholamban and phosphorylase a, used as substrates. The partially purified phosphatase could dephosphorylate the sites on phospholamban phosphorylated by either cAMP-dependent or calcium-calmodulin-dependent protein kinase(s). Enzymatic activity was inhibited by inhibitor-2 and by okadaic acid (I50 = 10-20 nM), using either phosphorylase a or phospholamban as substrates. The sensitivity of the phosphatase to inhibitor-2 or okadaic acid was similar for the two sites on phospholamban, phosphorylated by the cAMP-dependent and the calcium-calmodulin-dependent protein kinases. Phospholamban phosphatase activity was enhanced (40%) by Mg2+ or Mn2+ (3 mM) while Ca2+ (0.1-10 microM) had no effect. These characteristics suggest that the phosphatase associated with cardiac sarcoplasmic reticulum is a type 1 enzyme, and this activity may participate in the regulation of Ca2+ transport through dephosphorylation of phospholamban in cardiac muscle.
犬心肌肌浆网囊泡含有内在蛋白磷酸酶活性，该活性可使受磷蛋白去磷酸化并调节钙转运。有人提出这种磷酸酶是1型和2型酶的混合物（例如E.G.克拉尼亚斯和J.迪萨尔沃，1986年，《生物化学杂志》261卷，10029 - 10032页）。在本研究中，肌浆网磷酸酶活性用正辛基 - β - D - 葡萄糖苷溶解，并通过在DEAE - 葡聚糖凝胶、聚赖氨酸 - 琼脂糖、肝素 - 琼脂糖和DEAE - 葡聚糖凝胶上的连续层析进行纯化。从每根柱上洗脱下来的磷酸酶活性都只有一个峰，并且以受磷蛋白和磷酸化酶a作为底物时，洗脱峰是重合的。部分纯化的磷酸酶能够使受磷蛋白上被环磷酸腺苷（cAMP）依赖性或钙 - 钙调蛋白依赖性蛋白激酶磷酸化的位点去磷酸化。以磷酸化酶a或受磷蛋白作为底物时，酶活性受到抑制剂 - 2和冈田酸的抑制（半数抑制浓度I50 = 10 - 20 nM）。对于受磷蛋白上被cAMP依赖性和钙 - 钙调蛋白依赖性蛋白激酶磷酸化的两个位点，该磷酸酶对抑制剂 - 2或冈田酸的敏感性相似。Mg2 + 或Mn2 +（3 mM）可增强受磷蛋白磷酸酶活性（40%），而Ca2 +（0.1 - 10 microM）则无作用。这些特性表明，与心肌肌浆网相关的磷酸酶是1型酶，并且这种活性可能通过使心肌中的受磷蛋白去磷酸化参与钙转运的调节。
