Enhancement of rod outer segment GTPase accelerating protein activity by the inhibitory subunit of cGMP phosphodiesterase.
The cGMP phosphodiesterase (PDE) of retinal rod outer segments (ROS) is activated by the GTP-bound form of the G protein, transducin (Gt alpha). This activation can be reversed by the inhibitory gamma subunit of PDE through two distinct mechanisms: acceleration of GTP hydrolysis and direct inactivation independent of GTP hydrolysis. We have found that acceleration of Gt alpha GTPase by PDE gamma does not occur upon formation of a Gt alpha PDE gamma complex but rather reflects enhanced activity toward this complex of a membrane-bound GTPase accelerating protein. GTPase rate constants for Gt alpha in the presence of 3.3 microM PDE gamma were as high as 0.7 s-1 with hypotonically washed ROS membranes at 40 microM rhodopsin but were more than 10-fold lower when protein-free vesicles containing ROS lipids were substituted for ROS membranes. Acceleration of Gt alpha GTPase by PDE gamma was also barely detectable at low ROS concentrations (e.g. 4 microM rhodopsin) or if ROS treated with trypsin or urea were used. GTPase-independent inactivation by PDE gamma occurred efficiently at much lower membrane concentrations. Inhibition of Gt alpha-activated PDE was much slower than inhibition of PDE alpha beta by PDE gamma. Effects of PDE gamma upon successive additions of GTP suggested formation of a complex of PDE gamma and Gt alpha-GDP that is refractory to reactivation.
Angleson
J K
JK
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
Wensel
T G
TG
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000201:enzymology / Q000378:metabolism*; D015293:Transducin / Q000378:metabolism*
D008565:Membrane Proteins; D006160:Guanosine Triphosphate; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D020558:GTP Phosphohydrolases; D015293:Transducin