Modulation of the GTPase activity of transducin. Kinetic studies of reconstituted systems.
Modulation of the GTPase activity of transducin. Kinetic studies of reconstituted systems.
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转导蛋白 GTP 酶活性的调节。
作者：
A. Otto;B. Antonny;T. Vuong
我们试图确定视网膜cGMP磷酸二酯酶（PDE）对转导蛋白(T) GTPase活性的影响。一种新型的停流/快速过滤装置[Antonny， B., et . (1993) Biochemistry 32, 8646-8653]用于将不含棒状外段（ROS）膜的T - α - GTP输送到含有holoPDE的磷脂囊泡悬浮液中。通过pH电极测量，这些样品中不含其他蛋白质，只有T α和holoPDE， cGMP水解的衰变需要GTP水解，并在40秒内发生。向囊泡中加入T并不会加速这种失活。当ROS膜被尿素剥离，用转导蛋白+ holoPDE重建，并照亮时，注射一定量的GTP （holoPDE的亚化学计量量）会产生持续30秒的cGMP水解脉冲。然而，通过在等渗缓冲液中大量稀释来剥离活性氧进行相同的重构，其失活时间仅为8秒，与天然活性氧观察到的7秒相似。在这些等渗剥离的ROSs中，当第一次注射GTP γ S后再注射GTP时，cGMP水解脉冲延长，持续时间为17 S；尿素洗涤的活性氧未观察到这种延长。这些结果清楚地表明，holoPDE本身不能增强转导蛋白的GTPase活性，即使这两种蛋白定位在膜表面。相反，他们指出存在一种膜结合的尿素敏感蛋白因子，它可以激活转导素- holopde复合物中T α的GTPase。
We seek to define the influence of retinal cGMP phosphodiesterase (PDE) on the GTPase activity of transducin (T). A novel stopped-flow/fast filtration apparatus [Antonny, B., et al. (1993) Biochemistry 32, 8646-8653] is used to deliver T alpha GTP free of rod outer segment (ROS) membranes to a suspension of phospholipid vesicles bearing holoPDE. As measured by a pH electrode, the decay of cGMP hydrolysis from these samples, which contain no other proteins but T alpha and holoPDE, requires GTP hydrolysis and occurs in 40 s. The addition of T beta gamma to the vesicles does not accelerate this deactivation. When ROS membranes are urea-stripped, reconstituted with transducin + holoPDE, and illuminated, the injection of an amount of GTP that is substoichiometric to holoPDE gives a cGMP hydrolysis pulse that lasts for 30 s. However, the same reconstitution performed with ROS stripped by extensive dilution in isotonic buffer results in a deactivation time of only 8 s, which resembles the 7 s observed with native ROSs. With these isotonically stripped ROSs, when GTP injection comes after a first injection with GTP gamma S, the cGMP hydrolysis pulse is lengthened and lasts for 17 s; with urea-washed ROS, no such lengthening is observed. These results clearly demonstrate that holoPDE by itself cannot enhance the GTPase activity of transducin, even when the two proteins are localized on a membrane surface. Instead, they point to the existence of a membrane-bound, urea-sensitive protein factor that activates the GTPase of T alpha in the transducin-holoPDE complex.
牛杆外节转导蛋白 GTP 酶活性的调节。
DOI：
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发表时间：
1994
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Arshavsky,VY;Dumke,CL;Zhu,Y;Artemyev,NO;Skiba,NP;Hamm,HE;Bownds,MD
通讯作者：
Bownds,MD
杆外段 GTP 酶的增强通过 cGMP 磷酸二酯酶的抑制亚基加速蛋白质活性。
DOI：
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发表时间：
1994
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Angleson,JK;Wensel,TG
通讯作者：
Wensel,TG
GTP和Mg2对GTP酶活性和Go荧光特性的影响。
DOI：
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发表时间：
1987
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Higashijima,T;Ferguson,KM;Smigel,MD;Gilman,AG
通讯作者：
Gilman,AG