GTP hydrolysis by purified .alpha.-subunit of transducin and its complex with the cyclic GMP phosphodiesterase inhibitor

• Published in: Biochemistry (Easton) 1993-08, Vol.32 (33), p.8646-8653
• Main Authors: Antonny, Bruno, Otto-Bruc, Annie, Chabre, Marc, Minh Vuong, T
• Format: Article
• Language: English
• Subjects: 3',5'-Cyclic-GMP Phosphodiesterases - antagonists & inhibitors; 3',5'-Cyclic-GMP Phosphodiesterases - metabolism; Adenosine Triphosphate - metabolism; Animals; Biological and medical sciences; Cattle; Cell physiology; Cholera Toxin - metabolism; Filtration - instrumentation; Filtration - methods; Fundamental and applied biological sciences. Psychology; GTP Phosphohydrolases - metabolism; Guanosine 5'-O-(3-Thiotriphosphate) - metabolism; Guanosine Triphosphate - metabolism; Macromolecular Substances; Molecular and cellular biology; Rod Cell Outer Segment - metabolism; Signal transduction; Spectrometry, Fluorescence; Transducin - isolation & purification; Transducin - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00084a036

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.