Ho Y K, Fung B K
J Biol Chem. 1984 May 25;259(10):6694-9.
The properties and functions of the sulfhydryl groups of transducin were examined by 5,5' -dithiobis-(2-nitrobenzoic acid) titration and N-ethylmaleimide modification. The T beta gamma subunit of transducin contained a total of six free sulfhydryl groups and two were reactive under native conditions. Both reactive sulfhydryl groups were located in the beta polypeptide. The functions of transducin were not affected by the modification of these two sulfhydryl groups. The T alpha subunit of transducin contained three accessible sulfhydryl groups under both native and denaturing conditions. When 1.3 sulfhydryl groups were covalently modified by N-ethylmaleimide, the GTPase activity, the guanosine 5' -(beta, gamma-imido)triphosphate (Gpp(NH)p) uptake, and the rhodopsin-binding property of transducin were inhibited. The binding of Gpp(NH)p to T alpha blocked two of the three sulfhydryl groups from chemical modification and increased the reactivity of the remaining one. Modification of this specific sulfhydryl group of T alpha -Gpp(NH)p inhibited the exchange of the bound Gpp(NH)p for GTP. However, the modified T alpha-Gpp(NH)p was able to activate cGMP phosphodiesterase in solution and on positively charged liposomes. These findings demonstrated that a conformational change of T alpha occurs upon the binding of Gpp(NH)p and a specific sulfhydryl group of T alpha plays an important role in the activation of transducin in retinal rod outer segments.
通过5,5'-二硫代双（2-硝基苯甲酸）滴定法和N-乙基马来酰亚胺修饰法研究了转导蛋白巯基的性质和功能。转导蛋白的Tβγ亚基总共含有6个游离巯基，其中2个在天然条件下具有反应活性。两个具有反应活性的巯基均位于β多肽中。这两个巯基的修饰并未影响转导蛋白的功能。转导蛋白的Tα亚基在天然和变性条件下均含有3个可及的巯基。当1.3个巯基被N-乙基马来酰亚胺共价修饰后，转导蛋白的GTP酶活性、鸟苷5'-（β，γ-亚氨基）三磷酸（Gpp(NH)p）摄取以及视紫红质结合特性均受到抑制。Gpp(NH)p与Tα的结合使3个巯基中的2个免受化学修饰，并增加了剩余1个巯基的反应活性。Tα-Gpp(NH)p中这个特定巯基的修饰抑制了结合的Gpp(NH)p与GTP的交换。然而，修饰后的Tα-Gpp(NH)p能够在溶液中和带正电荷的脂质体上激活cGMP磷酸二酯酶。这些发现表明，Gpp(NH)p结合后Tα会发生构象变化，并且Tα的一个特定巯基在视网膜杆状细胞外段转导蛋白的激活中起重要作用。
