Pulvermüller A, Palczewski K, Hofmann K P
Institut für Biophysik und Strahlenbiologie, Universität Freiburg, Germany.
Biochemistry. 1993 Dec 28;32(51):14082-8. doi: 10.1021/bi00214a002.
Rhodopsin phosphorylation is a key event in the deactivation of this G-protein-coupled receptor. Rhodopsin kinase mediates the reaction and requires specific cytoplasmic loop domains on rhodopsin, distinct from the sites of phosphorylation, for binding and activation. In this study, we characterize the formation of a stable complex between photolyzed rhodopsin in native washed disk membranes and its kinase. Centrifugation of the membranes after illumination and subsequent polyacrylamide gel electrophoresis demonstrates light-dependent binding of rhodopsin kinase to the membranes. A real-time monitor for the transition of the solubilized kinase into the bound state is provided by flash-induced light-scattering binding signals. The complex has the following characteristics: (i) the on-rate of the reaction rises in linear proportion to the concentrations of both the kinase and photoactivated rhodopsin; kinetic analysis yields a bimolecular rate constant of kon = 0.5-1 microM-1s-1. (ii) The dissociation constant of the complex is 0.3 < KD < 0.5 microM in the absence of ATP, but with ATP, it decreases by at least a factor of 10; however, phosphorylation of rhodopsin or (auto)phosphorylation of rhodopsin kinase leads to destabilization of the complex. (iii) In contrast to the binding of arrestin and transducin, the binding of rhodopsin kinase to photoactivated rhodopsin does not stabilize the metarhodopsin II photoproduct; however, rhodopsin kinase competes with the G-protein transducin for binding to photoactivated rhodopsin. Extrapolation of the kinetic parameters to cellular concentrations at room temperature suggests that free competitive binding of the kinase would strongly inhibit the G-protein activation process after a few hundred catalytic cycles.
视紫红质磷酸化是该G蛋白偶联受体失活过程中的关键事件。视紫红质激酶介导此反应，并且需要视紫红质上特定的胞质环结构域（不同于磷酸化位点）用于结合和激活。在本研究中，我们表征了天然洗涤盘膜中光解视紫红质与其激酶之间稳定复合物的形成。光照后对膜进行离心，随后进行聚丙烯酰胺凝胶电泳，结果表明视紫红质激酶与膜存在光依赖性结合。闪光诱导的光散射结合信号提供了一种实时监测溶解的激酶向结合状态转变的方法。该复合物具有以下特征：（i）反应的结合速率与激酶和光活化视紫红质的浓度呈线性比例上升；动力学分析得出双分子结合速率常数kon = 0.5 - 1 μM-1s-1。（ii）在没有ATP的情况下，复合物的解离常数为0.3 < KD < 0.5 μM，但存在ATP时，解离常数至少降低10倍；然而，视紫红质的磷酸化或视紫红质激酶的（自）磷酸化会导致复合物不稳定。（iii）与抑制蛋白和转导蛋白的结合不同，视紫红质激酶与光活化视紫红质的结合不会稳定视紫红质II光产物；然而，视紫红质激酶与G蛋白转导蛋白竞争结合光活化视紫红质。将动力学参数外推至室温下的细胞浓度表明，激酶的自由竞争性结合在几百次催化循环后会强烈抑制G蛋白激活过程。
