Orr I, Shoshan-Barmatz V
Department of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel
Biochim Biophys Acta. 1996 Aug 14;1283(1):80-8. doi: 10.1016/0005-2736(96)00078-8.
This paper demonstrates and characterizes the inhibition of ryanodine binding caused by the phosphorylation of the 160/150-kDa proteins in skeletal muscle sarcoplasmic reticulum (SR). Inhibition of ryanodine binding was obtained by preincubation of SR membranes with ATP + NaF . The inhibition was characterized by the following findings: (a) If ATP was replaced by AdoPP[NH]P, inhibition of ryanodine binding activity was not observed. (b) The inhibitory effect of preincubation with ATP + NaF, like the phosphorylation of 150/160-kDa proteins, was Ca2+ dependent. (c) Inhibition of ryanodine binding, as the protein phosphorylation, was not observed if NaF (> 30 mM) was replaced with okadaic acid. (d) The optimal pH for the inhibition and the phosphorylation was about 7.0. (e) Both the phosphorylation of the 160/150-kDa proteins and inhibition of ryanodine binding were prevented by dichlorobenzimidazole riboside and hemin, inhibitors of casein kinase II. (f) Dephosphorylation of the 160/150-kDa proteins prevented the inhibition of ryanodine binding. (g) The presence of NP-40 during the phosphorylation prevented both the 160/150-kDa phosphorylation and the inhibition of ryanodine binding. Furthermore, a linear relationship was obtained between the degree of ryanodine binding inhibition and the level of phosphorylation of the 160/150-kDa proteins, as controlled by ATP or NaF concentrations. The binding affinity for Ca2+ of the ryanodine receptor (RyR) was modified by phosphorylation of the 160/150-kDa proteins, decreasing by up to 100-fold. The phosphorylation of the SR membranes resulted in an elimination of ryanodine binding sites with slight effect on the ryanodine binding affinity. These results suggest the modulation of the properties of the RyR by phosphorylation/dephosphorylation of the 160/150-kDa proteins. The identification of the phosphorylated 160/150-kDa proteins, their kinase, and the structural interactions between them and the RyR are presented in the accompanying paper.
本文展示并描述了骨骼肌肌浆网（SR）中160/150-kDa蛋白磷酸化对ryanodine结合的抑制作用。通过将SR膜与ATP + NaF预孵育来实现对ryanodine结合的抑制。该抑制作用具有以下特点：（a）若将ATP替换为AdoPP[NH]P，则未观察到ryanodine结合活性受到抑制。（b）与150/160-kDa蛋白的磷酸化一样，ATP + NaF预孵育的抑制作用依赖于Ca2+。（c）若将NaF（> 30 mM）替换为冈田酸，则未观察到ryanodine结合受到抑制，如同蛋白磷酸化情况一样。（d）抑制作用和磷酸化作用的最佳pH约为7.0。（e）160/150-kDa蛋白的磷酸化以及ryanodine结合的抑制均被酪蛋白激酶II的抑制剂二氯苯并咪唑核糖苷和血红素所阻止。（f）160/150-kDa蛋白的去磷酸化可防止ryanodine结合受到抑制。（g）磷酸化过程中NP-40的存在可同时阻止160/150-kDa蛋白的磷酸化以及ryanodine结合的抑制。此外，在ATP或NaF浓度的控制下，ryanodine结合抑制程度与160/150-kDa蛋白的磷酸化水平之间呈现线性关系。160/150-kDa蛋白的磷酸化使ryanodine受体（RyR）对Ca2+的结合亲和力发生改变，降低幅度高达100倍。SR膜的磷酸化导致ryanodine结合位点的消除，对ryanodine结合亲和力影响较小。这些结果表明，160/150-kDa蛋白的磷酸化/去磷酸化可调节RyR的特性。随附论文介绍了磷酸化的160/150-kDa蛋白的鉴定、其激酶以及它们与RyR之间的结构相互作用。
