Regulation of the cGMP phosphodiesterase in bovine rod outer segments. Use of resonance energy transfer to distinguish between associative and dissociative activation mechanisms.
Resonance energy transfer was used to monitor the interactions of the gamma subunit of the cyclic GMP phosphodiesterase (gamma PDE) with the alpha and beta subunits, which together comprise the catalytic core of the enzyme (alpha beta PDE). An iodoacetamidofluorescein (IAF)-labeled alpha beta PDE (IAF-PDEt) served as the acceptor species in these experiments while a coumarin maleimide-modified gamma PDE (CPM-gamma PDE) served as the fluorescent donor. Trypsin treatment of an IAF-labeled, intact phosphodiesterase (PDE) complex resulted in the selective proteolysis of the gamma PDE and full activation of enzyme activity. The binding of IAF-PDEt to the CPM-gamma PDE was monitored directly by measuring the quenching (25-35%) of the CPM fluorescence emission or by measuring the changes in the rate cGMP hydrolysis by IAF-PDEt using the pH electrode method. Both methods yielded titration curves which were well fitted using a single affinity constant (Kd approximately 1-2 nM). Under conditions where added CPM-gamma PDE resulted in complete inhibition of the IAF-PDEt activity and, conversely, a maximal quenching of the CPM-gamma PDE fluorescence by added IAF-PDEt, the subsequent addition of guanosine 5'-O-(thiotriphosphate) bound form of the alpha subunit of transducin (alpha TGTP gamma S) restored approximately 50% of the enzyme activity while restoring < 30% of the CPM-gamma PDE fluorescence emission. Based on these results, we suggest that a portion of the observed PDE activation by alpha TGTP gamma S occurs via an associated complex of alpha TGTP gamma S and the enzyme.
Department of Pharmacology, Cornell University, Ithaca, New York 14853-6401.
J W
JW
Erickson
R A
RA
Cerione
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D004735:Energy Transfer; D004789:Enzyme Activation; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006706:Homeostasis; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D005452:Fluoresceins; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; C015296:5-iodoacetamidofluorescein; D015106:3',5'-Cyclic-GMP Phosphodiesterases