Runnels L W, Scarlata S F
Department of Physiology & Biophysics, State University of New York at Stony Brook 11794-8661, USA.
Biochemistry. 1998 Nov 3;37(44):15563-74. doi: 10.1021/bi9811258.
The activity of mammalian phosphoinositide-specific phospholipase C beta 2 (PLC-beta 2) is regulated by the alpha q family of G proteins and by beta gamma subunits. We measured the affinity between the laterally associating PLC-beta 2 and G beta gamma on membrane surfaces by fluorescence resonance energy transfer. Using a simple model, we translated this apparent affinity to a bulk or three-dimensional equilibrium constant (Kd) and obtained a value of 3.2 microM. We confirmed this Kd by separately measuring the on and off (kf and kr) rate constants. The kf was slower than a diffusion-limited value, suggesting that conformational changes occur when the two proteins interact. The off rate shows that the PLC-beta 2.G beta gamma complexes are long-lived ( approximately 123 s) and that activation of PLC-beta 2 by G beta gamma would be sustained without a deactivating factor. The addition of alpha i1(GDP) subunits failed to physically dissociate the complex as determined by fluorescence. However, enzyme activity studies performed under similar conditions show that the addition of G alpha i1(GDP) results in reversal of PLC-beta 2 activation by G beta gamma during the time of the assay (30 s). From these results, we propose that G alpha(GDP) subunits can bind to the PLC-beta 2.G beta gamma complex to allow for rapid deactivation without complex dissociation. In support of this model, we show by fluorescence that G alpha i1(GDP).G beta gamma.PLC-beta 2 can form.
哺乳动物磷酸肌醇特异性磷脂酶Cβ2（PLC-β2）的活性受G蛋白的αq家族和βγ亚基调控。我们通过荧光共振能量转移测量了膜表面横向结合的PLC-β2与Gβγ之间的亲和力。使用一个简单模型，我们将这种表观亲和力转化为整体或三维平衡常数（Kd），得到的值为3.2微摩尔。我们通过分别测量结合和解离（kf和kr）速率常数来确认这个Kd。kf低于扩散限制值，表明两种蛋白质相互作用时发生了构象变化。解离速率表明PLC-β2·Gβγ复合物寿命较长（约123秒），并且在没有失活因子的情况下，Gβγ对PLC-β2的激活将持续。荧光检测结果表明，添加αi1（GDP）亚基未能使复合物物理解离。然而，在类似条件下进行的酶活性研究表明，添加Gαi1（GDP）会在检测期间（30秒）导致Gβγ对PLC-β2的激活逆转。基于这些结果，我们提出Gα（GDP）亚基可以结合到PLC-β2·Gβγ复合物上，从而在不解离复合物的情况下实现快速失活。为支持该模型，我们通过荧光显示Gαi1（GDP）·Gβγ·PLC-β2可以形成。
