Wilden U, Hall S W, Kühn H
Proc Natl Acad Sci U S A. 1986 Mar;83(5):1174-8. doi: 10.1073/pnas.83.5.1174.
Each photoexcited rhodopsin (R*) molecule catalyzes binding of GTP to many copies of the guanine nucleotide-binding protein transducin, which, in its GTP-binding form, then activates cGMP phosphodiesterase (PDEase). Subsequent deactivation of this light-activated enzyme cascade involves hydrolysis of the GTP bound to transducin, as well as decay of the activating capacity of R*. We report here that deactivation of PDEase in rod outer segment suspensions is highly enhanced by addition of ATP and purified 48-kDa protein, which is an intrinsic rod outer segment protein that is soluble in the dark but binds to photolyzed rhodopsin that has been phosphorylated by rhodopsin kinase and ATP [Kühn, H., Hall, S.W. & Wilden, U. (1984) FEBS Lett. 176, 473-478]. To analyze the mechanism by which ATP and 48-kDa protein deactivate PDEase, we used an ATP-free system consisting of thoroughly washed disk membranes, whose rhodopsin had been previously phosphorylated and chromophore-regenerated, and to which purified PDEase and transducin were reassociated. Such phosphorylated membranes exhibited a significantly lower (by a factor less than or equal to 5) light-induced PDEase-activating capacity than unphosphorylated controls. Addition of purified 48-kDa protein to phosphorylated membranes further suppressed their PDEase-activating capacity; suppression could be as high as 98% (as compared to unphosphorylated membranes), depending on the amount of 48-kDa protein and the flash intensity. Addition of ATP had little further effect. In contrast, PDEase activation or deactivation with unphosphorylated control membranes was not influenced by 48-kDa protein, even in the presence of ATP, provided rhodopsin kinase was absent. Our data suggest that 48-kDa protein binds to phosphorylated R* and thereby quenches its capacity to activate transducin and PDEase.
每个光激发的视紫红质（R*）分子催化鸟嘌呤核苷酸结合蛋白转导蛋白的多个拷贝与GTP结合，转导蛋白以其GTP结合形式随后激活cGMP磷酸二酯酶（PDEase）。这种光激活酶级联反应的后续失活涉及与转导蛋白结合的GTP的水解，以及R激活能力的衰减。我们在此报告，在视杆细胞外段悬浮液中加入ATP和纯化的48 kDa蛋白可显著增强PDEase的失活，48 kDa蛋白是视杆细胞外段的一种内在蛋白，在黑暗中可溶，但可与已被视紫红质激酶和ATP磷酸化的光解视紫红质结合[库恩，H.，霍尔，S.W. & 威尔登，U.（1984年）《欧洲生物化学学会联合会快报》176, 473 - 478]。为了分析ATP和48 kDa蛋白使PDEase失活的机制，我们使用了一个无ATP系统，该系统由彻底洗涤过的盘状膜组成，其视紫红质先前已被磷酸化并进行了发色团再生，纯化的PDEase和转导蛋白重新与之结合。与未磷酸化的对照相比，这种磷酸化的膜表现出显著更低（小于或等于5倍）的光诱导PDEase激活能力。向磷酸化的膜中加入纯化的48 kDa蛋白进一步抑制了它们的PDEase激活能力；抑制程度可达98%（与未磷酸化的膜相比），这取决于48 kDa蛋白的量和闪光强度。加入ATP几乎没有进一步的影响。相反，即使存在ATP且视紫红质激酶不存在，未磷酸化的对照膜的PDEase激活或失活也不受48 kDa蛋白的影响。我们的数据表明，48 kDa蛋白与磷酸化的R结合，从而淬灭其激活转导蛋白和PDEase的能力。
