Each injection was followed by a buffer flow for ~7 min to monitor the dissociation of the complex. For kinetics studies 35 μl of varying concentrations of G tαGTPγS or trPDE αβ were injected at a flow rate of 5 μl/min in a buffer containing 120 mM NaCl, 8 mM MgCl 2, 1 mM dithiothreitol, 0.05 mg/ml bovine serum albumin, and 10 mM HEPES-KOH (pH 7.5). The noncovalently bound PDE γ was then removed by a 5-μl pulse of 6 M guanidine, 100 mM NaCl, 1 mM dithiothreitol, and 10 mM Tris-HCl (pH 8.0). Unbound groups were blocked by 30 μl of 1 M ethanolamine (pH 8.5). Briefly, the CM5 chip was activated at the flow rate of 5 μl/min with 30 μl of 0.2 M N-(3-dimethylaminopropyl)- N-ethylcarbodiimide and 0.4 M of N-hydroxysuccinimide, and then 15-45 μl of 0.5 μM PDE γ in 100 mM NaCl with 10 mM sodium formate (pH 4.3) were flown through the activated surface.
PDE γ was covalently attached to the surface of the BIAcore sensor chip (Pharmacia Biosensor) via primary amines following the activation of the carboxymethyl groups of dextran on the chip. These observations suggest that the role of PDE γ in activation of the transducin GTPase rate may be based on multiple hydrophobic interactions between these molecules. Single substitutions of 7 other hydrophobic amino acids resulted in a 50-70% reduction in the ability of PDE γ to stimulate transducin GTPase, while substitutions of charged and polar amino acids had little or no effect. This mutation also resulted in a reduction of PDE γ affinity for transducin, but did not affect PDE γ interactions with the phosphodiesterase catalytic subunits. The substitution of tryptophan at position 70 completely abolished the acceleration of GTP hydrolysis by transducin in a complex with this mutant. Here we characterize the stimulation of transducin GTPase by a set of 23 mutant phosphodiesterase γ-subunits (PDE γ) containing single alanine substitutions within a stretch of the 25 C-terminal amino acid residues known to be primarily responsible for the GTPase regulation. In the phototransduction cascade the GTPase activity of photoreceptor G-protein, transducin, is substantially accelerated in a complex with its effector, cGMP phosphodiesterase. In several signal transduction pathways, including the cGMP cascade of photoreceptor cells, the relatively slow GTPase activity of heterotrimeric G-proteins can be significantly accelerated when they are complexed with corresponding effectors. α-Subunits then modulate the activity of downstream effectors until the bound GTP is hydrolyzed. G-protein interactions with activated receptors catalyze the replacement of GDP bound to the α-subunit with GTP. Heterotrimeric G-proteins mediate between receptors and effectors, acting as molecular clocks. Glycobiology and Extracellular Matrices.
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