GTP hydrolysis by purified alpha-subunit of transducin and its complex with the cyclic GMP phosphodiesterase inhibitor.
The single-turn GTP hydrolysis by isolated and soluble transducin has been time-resolved using a rapid flow filtration technique which takes advantage of the GTP-requiring detachment of transducin alpha-subunits (T alpha) from photoactivated rhodopsin (R*). Illuminated rod outer segment (ROS) fragments to which holo-transducin is tightly bound are retained on a syringe filter that is washed continuously with a buffer containing no GTP. When the flow is switched to a buffer with GTP, T alpha GTP is specifically eluted and injected into a cuvette where GTP hydrolysis is monitored via the associated change in the T alpha intrinsic tryptophan fluorescence. Low concentrations of GTP elute the complete pool of T alpha from the filter-retained ROS fragments in less than 1 s. This directly demonstrates that, upon GTP loading, T alpha becomes instantly soluble in physiological buffers (120 mM KC1 and 2 mM MgCl2). When all alone, T alpha hydrolyzes its bound GTP in 21 +/- 1 s (1/e time at 25 degrees C). Replacing chloride by other anions increases the GTPase rate by 2-fold. The K50 for chloride inhibition of GTPase is approximately 2 mM. Slower GTP hydrolysis is observed for cholera-toxin-modified transducin or when GTP alpha S (Sp) replaces GTP in the eluting buffer. No signal is observed when GTP gamma S is used. The GTPase rate is unaffected when T alpha GTP binds to the inhibitory subunit (PDE gamma) of the cGMP phosphodiesterase (PDE), although this binding is fast and of high affinity.(ABSTRACT TRUNCATED AT 250 WORDS)
Centre National de la Recherche Scientifique, Institut de Pharmacologie Moléculaire et Cellulaire, Yalbonne, France.
B
B
Antonny
A
A
Otto-Bruc
M
M
Chabre
T M
TM
Vuong
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002772:Cholera Toxin / Q000378:metabolism; D005374:Filtration / Q000295:instrumentation / Q000379:methods; D020558:GTP Phosphohydrolases / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D046911:Macromolecular Substances; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D015293:Transducin / Q000302:isolation & purification / Q000378:metabolism*
D046911:Macromolecular Substances; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D002772:Cholera Toxin; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D020558:GTP Phosphohydrolases; D015293:Transducin