Yamazaki A, Bondarenko V A, Dua S, Yamazaki M, Usukura J, Hayashi F
Kresge Eye Institute, Wayne State University, School of Medicine, Detroit, Michigan 48201, USA.
J Biol Chem. 1996 Dec 20;271(51):32495-8. doi: 10.1074/jbc.271.51.32495.
Cyclic GMP phosphodiesterase, a key enzyme for phototransduction, contains alpha, beta (Palphabeta), and two gamma (Pgamma) subunits. In addition to catalytic sites, Palphabeta has two classes of noncatalytic cGMP binding sites with different affinities (Kd values <100 nM and >1 microM). Pgamma regulates Palphabeta as an inhibitor of cGMP hydrolysis and as a stimulator of cGMP binding to the high affinity noncatalytic sites. Pgamma release from Palphabeta by the GTP-bound alpha subunit of transducin (GTP.Talpha) interrupts these two functions. Here we describe a novel regulation of the Pgamma release by [cGMP] and its physiological implication. We isolated Pgamma mutants that exhibit abnormally one of these two functions, indicating the distinct domains in Pgamma are involved to express these functions. When [cGMP] was high ( approximately 5 microM), Pgamma responsible for the inhibition of cGMP hydrolysis was preferentially released, and cGMP hydrolysis activity of Palphabeta was increased about 10 times. When [cGMP] was low (less than approximately 0.5 microM), Pgamma responsible for the stimulation of cGMP binding to the high affinity sites was released. The Pgamma release resulted in the decrease of relative affinity of cGMP for the high affinity sites to at least (null)/1;10, followed by the rapid release of cGMP from one of the high affinity sites (apparent t1/2 = 3.8 s). cGMP ( approximately 5 microM) inhibited the extraction of Palphabeta from rod membranes by a Mg2+-free hypotonic buffer. The inhibition of Palphabeta extraction was not affected by Pgamma, suggesting that Palphabeta detects on the order of micromolar [cGMP] using low affinity noncatalytic sites on Palphabeta. Because [cGMP] is approximately 5 microM in darkness and lowered by photoexcitation and phosphodiesterase concentration is approximately 30 microM in rod photoreceptors, it is possible that cGMP phosphodiesterase functions to increase cytoplasamic [cGMP] after [cGMP] is reduced to the illuminated level.
环鸟苷酸磷酸二酯酶是光转导的关键酶，由α、β（Palphabeta）和两个γ（Pgamma）亚基组成。除催化位点外，Palphabeta有两类具有不同亲和力（解离常数Kd值<100 nM和>1 μM）的非催化性环鸟苷酸结合位点。Pgamma作为环鸟苷酸水解的抑制剂和环鸟苷酸与高亲和力非催化位点结合的刺激剂来调节Palphabeta。转导素的GTP结合α亚基（GTP.Talpha）使Pgamma从Palphabeta释放，从而中断这两种功能。在此我们描述了由[cGMP]对Pgamma释放的一种新调节及其生理意义。我们分离出了表现出这两种功能之一异常的Pgamma突变体，表明Pgamma中不同的结构域参与表达这些功能。当[cGMP]较高（约5 μM）时，负责抑制环鸟苷酸水解的Pgamma优先释放，Palphabeta的环鸟苷酸水解活性增加约10倍。当[cGMP]较低（小于约0.5 μM）时，负责刺激环鸟苷酸与高亲和力位点结合的Pgamma释放。Pgamma的释放导致环鸟苷酸对高亲和力位点的相对亲和力降低至至少1/10，随后环鸟苷酸从其中一个高亲和力位点快速释放（表观半衰期t1/2 = 3.8秒）。环鸟苷酸（约5 μM）抑制无镁低渗缓冲液从视杆细胞膜中提取Palphabeta。Palphabeta提取的抑制不受Pgamma影响，这表明Palphabeta利用Palphabeta上的低亲和力非催化位点检测微摩尔级别的[cGMP]。由于在黑暗中环鸟苷酸浓度约为5 μM，光照使其降低，且视杆光感受器中环鸟苷酸磷酸二酯酶浓度约为30 μM，所以有可能在环鸟苷酸降低到光照水平后，环鸟苷酸磷酸二酯酶起到增加细胞质中环鸟苷酸浓度的作用。
