Hawkins C, Xu A, Narayanan N
Department of Physiology, University of Western Ontario, London, Canada.
Mol Cell Biochem. 1995 Jan 26;142(2):131-8. doi: 10.1007/BF00928934.
In both cardiac and slow-twitch skeletal muscle sarcoplasmic reticulum (SR) there are several systems involved in the regulation of Ca(2+)-ATPase function. These include substrate level regulation, covalent modification via phosphorylation-dephosphorylation of phospholamban by both cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase (CaM kinase) as well as direct CaM kinase phosphorylation of the Ca(2+)-ATPase. Studies comparing the effects of PKA and CaM kinase on cardiac Ca(2+)-ATPase function have yielded differing results; similar studies have not been performed in slow-twitch skeletal muscle. It has been suggested recently, however, that phospholamban is not tightly coupled to the Ca(2+)-ATPase in SR vesicles from slow-twitch skeletal muscle. Our results indicate that assay conditions strongly influence the extent of CaM kinase-dependent Ca(2+)-ATPase stimulation seen in both cardiac and slow-twitch skeletal muscle. Addition of calmodulin (0.2 microM) directly to the Ca2+ transport assay medium results in minimal (approximately 112-130% of control) stimulation of Ca2+ uptake activity when the Ca2+ uptake reaction is initiated by the addition or either ATP or Ca2+/EGTA. On the other hand, prephosphorylation of the SR by the endogenous CaM kinase and subsequent transfer of the membranes to the Ca2+ transport assay medium results in stimulation of Ca2+ uptake activity (202% of control). These effects are observable in both cardiac and slow-twitch skeletal muscle SR. PKA stimulates Ca2+ uptake markedly (215% of control) when the Ca2+ uptake reaction is initiated by the addition of prephosphorylated SR membranes or by Ca2+/EGTA but minimally (130% of control) when the Ca2+ uptake reaction is initiated by the addition of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)
在心肌和慢肌纤维骨骼肌的肌浆网（SR）中，有几个系统参与钙（Ca2+）-ATP酶功能的调节。这些系统包括底物水平调节、通过环磷腺苷（cAMP）依赖性蛋白激酶（PKA）和钙/钙调蛋白依赖性蛋白激酶（CaM激酶）对受磷蛋白进行磷酸化-去磷酸化的共价修饰，以及CaM激酶对Ca2+-ATP酶的直接磷酸化。比较PKA和CaM激酶对心肌Ca2+-ATP酶功能影响的研究得出了不同的结果；类似的研究尚未在慢肌纤维骨骼肌中进行。然而，最近有人提出，在慢肌纤维骨骼肌的SR囊泡中，受磷蛋白与Ca2+-ATP酶的耦合并不紧密。我们的结果表明，测定条件强烈影响在心肌和慢肌纤维骨骼肌中观察到的CaM激酶依赖性Ca2+-ATP酶刺激程度。当通过添加ATP或Ca2+/乙二醇双四乙酸（EGTA）启动Ca2+摄取反应时，直接向Ca2+转运测定培养基中添加钙调蛋白（0.2微摩尔）会导致Ca2+摄取活性的最小刺激（约为对照的112%-130%）。另一方面，内源性CaM激酶对SR进行预磷酸化，随后将膜转移到Ca2+转运测定培养基中，会导致Ca2+摄取活性的刺激（为对照的202%）。这些效应在心肌和慢肌纤维骨骼肌的SR中均可观察到。当通过添加预磷酸化的SR膜或Ca2+/EGTA启动Ca2+摄取反应时，PKA会显著刺激Ca2+摄取（为对照的2
