GUANINE-NUCLEOTIDE BINDING CHARACTERISTICS OF TRANSDUCIN - ESSENTIAL ROLE OF RHODOPSIN FOR RAPID EXCHANGE OF GUANINE-NUCLEOTIDES
GUANINE-NUCLEOTIDE BINDING CHARACTERISTICS OF TRANSDUCIN - ESSENTIAL ROLE OF RHODOPSIN FOR RAPID EXCHANGE OF GUANINE-NUCLEOTIDES
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DOI：
10.1021/bi00467a030
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发表时间：
1990-04-17
期刊：
影响因子：
2.9
通讯作者：
NORTHUP, JK
中科院分区：
文献类型：
--
作者：
FAWZI, AB;NORTHUP, JK
转导蛋白（Transducin，Gt）是受体偶联信号转导鸟嘌呤核苷酸（guanine monucleotide，GN）结合蛋白（G蛋白）家族的成员。已知光活化视紫红质催化Gt上的GN交换，导致形成Gt α-葡萄糖的活性状态。GTP复合物。然而，已显示纯化的Gt制剂在不存在活化受体的情况下交换GN [Wessling-Resnick，M.，和约翰逊，G. L.（1987）Biochemistry 26，4316-4323]。为了评价视紫红质在Gt活化中的作用，我们研究了不同Gt制剂的GN结合特性。Gt制剂得自GTP处理的牛视杆外节（ROS）盘的上清液，随后在Sephadex G-25柱上除去游离GTP，在30 ℃下结合GTP γ S。C中，在没有加入外源性视紫红质的情况下，活性为1摩尔GTP γ S结合/摩尔Gt（Gt-I制剂）。GTP γ S与Gt-I制剂的结合与ROS盘cGMP磷酸二酯酶的活化密切相关。Gt-I制剂的GN-结合活性依赖于反应温度，在4 ℃下没有观察到结合。C.在10 μ M漂白视紫红质存在下，Gt-I制剂在4 ℃结合GTP γ S。C.然而，Gt-I制剂的己基琼脂糖层析导致Gt制剂在30 ℃温育60分钟后显示<0.1mol/mol的结合活性。C在没有视紫红质的情况下（Gt-II制剂）。在低至0.03 μ M的漂白视紫红质存在下，Gt-II制剂在30 ℃快速结合GTP γ S。C. Gt-I和Gt-II制剂的等摩尔混合物显示出1.0 mol/mol的结合活性，在没有添加视紫红质。虽然用羟胺处理Gt-I制剂（已知羟胺将视紫红质转化为视蛋白）大大降低了GTP γ S与制剂结合的速率，但在除去羟胺后加入0.03 μ M漂白视紫红质恢复了结合活性。这些结果表明：（1）视紫红质对于Gt上的快速GN交换是必需的;（2）在某些Gt制剂中观察到的在不存在外源性视紫红质的情况下的快速GN交换受到未检测到的视紫红质污染的刺激。
Transducin (Gt) is a member of a family of receptor-coupled signal-transducing guanine mucleotide (GN) binding proteins (G-proteins). Light-activated rhodopsin is known to catalyze GN exchange on Gt, resulting in the formation of the active state of the Gt.alpha.-GTP complex. However, purified preparations of Gt have been shown to exchange GN in the absence of activated receptors [Wessling-Resnick, M., and Johnson, G. L. (1987) Biochemistry 26, 4316-4323]. To evaluate the role of rhodopsin in the activation of Gt, we studied GN-binding characteristics of different preparations of Gt. Gt preparations obtained from the supernate of GTP-treated bovine rod outer segment (ROS) disks, followed by removal of free GTP on a Sephadex G-25 column, bound GTP.gamma.S at 30.degree. C in the absence of added exogenous rhodopsin with an activity of 1 mol of GTP.gamma.S bound/mol of Gt (Gt-I preparations). Binding of GTP.gamma.S to Gt-I preparations closely correlated with the activation of ROS disk cGMP phosphodiesterase. GN-binding activity of Gt-I preparations was dependent on reaction temperature, and no binding was observed at 4.degree. C. In the presence of 10 .mu.M bleached rhodopsin, Gt-I preparations bound GTP.gamma.S at 4.degree. C. However, hexylagarose chromatography of Gt-I preparations led to a preparation of Gt that showed < 0.1 mol/mol binding activity following 60-min incubation at 30.degree. C in the absence of rhodopsin (Gt-II preparations). In the presence of as low as 0.03 .mu.M bleached rhodopsin, Gt-II preparations rapidly bound GTP.gamma.S at 30.degree. C. Equimolar mixtures of Gt-I and Gt-II preparations showed a 1.0 mol/mol binding activity in the absence of added rhodopsin. While treatment of Gt-I preparations with hydroxylamine, which is known to convert rhodopsin to opsin, greatly diminished the rate of GTP.gamma.S binding to the preparation, addition of 0.03 .mu.M bleached rhodopsin following removal of hydroxylamine restored the binding activity. These results indicate that (1) rhodopsin is essential for rapid GN exchange on Gt and (2) rapid GN exchange in the absence of exogenous rhodopsin observed in some Gt preparations is stimulated by undetected rhodopsin contamination.