Yoshida Y, Cai J Q, Imai S
Department of Pharmacology, Niigata University School of Medicine.
J Biochem. 1992 May;111(5):559-62. doi: 10.1093/oxfordjournals.jbchem.a123796.
A plasma membrane Ca(2+)-pump ATPase preparation purified from porcine aorta was incubated with cGMP-dependent protein kinase (G-kinase) under the conditions under which dose-dependent stimulation of the enzyme by G-kinase was observed. Several proteins were phosphorylated, but two isoforms of plasma membrane Ca(2+)-pump ATPase with molecular masses of 135- and 145-kDa were not phosphorylated. The protein that was phosphorylated by G-kinase and identified in our previous study as the 135-kDa isoform of Ca(2+)-pump ATPase, on the basis of its almost identical mobility on SDS-PAGE, was found to be another protein with a molecular mass of 138 kDa. Fractionation of the enzyme preparation after incubation with G-kinase by a newly developed calmodulin affinity chromatographic method resulted in the separation of all the G-kinase substrates from the two isoforms of plasma membrane Ca(2+)-pump ATPase. These results suggest that the direct phosphorylation of the Ca(2+)-pump ATPase does not occur in association with the stimulation of the plasma membrane Ca(2+)-pump ATPase by G-kinase.
将从猪主动脉中纯化得到的质膜Ca(2+)-泵ATP酶制剂，在能观察到G激酶对该酶产生剂量依赖性刺激作用的条件下，与环磷酸鸟苷依赖性蛋白激酶（G激酶）一起孵育。有几种蛋白质发生了磷酸化，但分子量分别为135 kDa和145 kDa的两种质膜Ca(2+)-泵ATP酶同工型未发生磷酸化。在我们之前的研究中，根据其在SDS-PAGE上几乎相同的迁移率，被鉴定为Ca(2+)-泵ATP酶135 kDa同工型的、被G激酶磷酸化的蛋白质，实际上是另一种分子量为138 kDa的蛋白质。采用新开发的钙调蛋白亲和色谱法，对与G激酶孵育后的酶制剂进行分级分离，结果所有G激酶底物都与质膜Ca(2+)-泵ATP酶的两种同工型分离开来。这些结果表明，质膜Ca(2+)-泵ATP酶的直接磷酸化并不伴随G激酶对质膜Ca(2+)-泵ATP酶的刺激作用而发生。
