Kwok-Keung Fung B, Stryer L
Proc Natl Acad Sci U S A. 1980 May;77(5):2500-4. doi: 10.1073/pnas.77.5.2500.
We have studied the binding of guanyl nucleotides to retinal rod outer segment membranes to determine how light activates a cyclic GMP phosphodiesterase and a GTPase. We found that rod outer segment membranes contain tightly bound radioactive GDP after incubation in the dark with [3H]GDP or [alpha-32P]GTP. Reconstituted membranes containing only rhodopsin and phospholipid bind almost no GDP. More than 80% of the radioactive GDP bound to rod outer segment membranes could be released by subsequent illumination. At low light levels, the rate and extent of GDP release were markedly enhanced by the presence of GTP or p[NH]ppG, a nonhydrolyzable analog of GTP. The kinetics of binding of p[NH]ppG paralleled the kinetics of release of bound GDP, indicating that p[NH]ppG was exchanged for bound GDP. The maximal amount of bound p[NH]ppG was 1 per 30 rhodopsins when photolyzed membranes were incubated with 10 micro M nucleotide. Under these conditions, p[NH]ppG binding was half-maximal when only 1 in 90,000 rhodopsins was photolyzed. This corresponds to the catalyzed exchange of 500 p[NH]ppG for bound GDP per photolyzed rhodopsin. We propose a light-activated GTP-GDP amplification cycle involving a guanyl nucleotide binding protein with GTPase activity (E). The essence of this cycle is that photolyzed rhodopsin catalyzes the formation of E . GTP from E . GDP (the major species in the dark) by nucleotide exchange. The formation of several hundred E . GTP per photolyzed rhodopsin may be the first stage of amplification in visual excitation.
我们研究了鸟苷酸与视网膜视杆细胞外段膜的结合情况，以确定光如何激活环鸟苷酸磷酸二酯酶和一种GTP酶。我们发现，在黑暗中用[³H]GDP或[α-³²P]GTP孵育后，视杆细胞外段膜含有紧密结合的放射性GDP。仅含有视紫红质和磷脂的重构膜几乎不结合GDP。与视杆细胞外段膜结合的放射性GDP中，超过80%可通过随后的光照释放。在低光照水平下，GTP或p[NH]ppG（一种不可水解的GTP类似物）的存在显著增强了GDP释放的速率和程度。p[NH]ppG的结合动力学与结合的GDP释放动力学平行，表明p[NH]ppG与结合的GDP发生了交换。当用10微摩尔核苷酸孵育光解的膜时，结合的p[NH]ppG的最大量为每30个视紫红质1个。在这些条件下，当每90,000个视紫红质中只有1个被光解时，p[NH]ppG的结合量达到最大值的一半。这相当于每个光解的视紫红质催化500个p[NH]ppG与结合的GDP进行交换。我们提出了一个光激活的GTP - GDP放大循环，涉及一种具有GTP酶活性的鸟苷酸结合蛋白（E）。这个循环的本质是光解的视紫红质通过核苷酸交换催化从E·GDP（黑暗中的主要形式）形成E·GTP。每个光解的视紫红质形成数百个E·GTP可能是视觉兴奋放大的第一阶段。
