Cook T A, Ghomashchi F, Gelb M H, Florio S K, Beavo J A
Department of Pharmacology, University of Washington, Seattle, WA 98195, USA.
J Biol Chem. 2001 Feb 16;276(7):5248-55. doi: 10.1074/jbc.M004690200. Epub 2000 Oct 26.
The delta subunit of the rod photoreceptor PDE has previously been shown to copurify with the soluble form of the enzyme and to solubilize the membrane-bound form (). To determine the physiological effect of the delta subunit on the light response of bovine rod outer segments, we measured the real time accumulation of the products of cGMP hydrolysis in a preparation of permeablized rod outer segments. The addition of delta subunit GST fusion protein (delta-GST) to this preparation caused a reduction in the maximal rate of cGMP hydrolysis in response to light. The maximal reduction of the light response was about 80%, and the half-maximal effect occurred at 385 nm delta subunit. Several experiments suggest that this effect was not due to the effects of delta-GST on transducin or rhodopsin kinase. Immunoblots demonstrated that exogenous delta-GST solubilized the majority of the PDE in ROS but did not affect the solubility of transducin. Therefore, changes in the solubility of transducin cannot account for the effects of delta-GST in the pH assay. The reduction in cGMP hydrolysis was independent of ATP, which indicates that it was not due to effects of delta-GST on rhodopsin kinase. In addition to the effect on cGMP hydrolysis, the delta-GST fusion protein slowed the turn-off of the system. This is probably due, at least in part, to an observed reduction in the GTPase rate of transducin in the presence of delta-GST. These results demonstrate that delta-GST can modify the activity of the phototransduction cascade in preparations of broken rod outer segments, probably due to a functional uncoupling of the transducin to PDE step of the signal transduction cascade and suggest that the delta subunit may play a similar role in the intact outer segment.
视杆光感受器磷酸二酯酶（PDE）的δ亚基先前已被证明可与该酶的可溶性形式共同纯化，并使膜结合形式溶解（）。为了确定δ亚基对牛视杆外段光反应的生理作用，我们在通透的视杆外段制剂中测量了cGMP水解产物的实时积累。向该制剂中添加δ亚基GST融合蛋白（δ-GST）导致光刺激下cGMP水解的最大速率降低。光反应的最大降低约为80%，半最大效应出现在385 nM的δ亚基处。多项实验表明，这种效应不是由于δ-GST对转导素或视紫红质激酶的影响。免疫印迹显示，外源性δ-GST使ROS中的大部分PDE溶解，但不影响转导素的溶解度。因此，转导素溶解度的变化不能解释δ-GST在pH测定中的作用。cGMP水解的降低与ATP无关，这表明它不是由于δ-GST对视紫红质激酶的影响。除了对cGMP水解的影响外，δ-GST融合蛋白还减缓了系统的关闭。这可能至少部分是由于在存在δ-GST的情况下观察到转导素的GTPase速率降低。这些结果表明，δ-GST可以改变破碎视杆外段制剂中光转导级联的活性，这可能是由于转导素与信号转导级联的PDE步骤功能解偶联所致，并表明δ亚基可能在完整的外段中发挥类似作用。
