Effect of detergents and lipids on transducin photoactivation by rhodopsin

Rhodopsin samples, isolated using four different extraction procedures, were used to investigate the photodependent activation of the GTPase activity of transducin. A complete inhibition of transducin light-dependent GTP hydrolytic activity was observed when rhodopsin purified in the presence of 1 per cent digitonin, following rod outer segment (ROS) solubilization with 1 per cent 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate (CHAPS), WAS used for its activation [0 pmol of inorganic phosphate (Pi) released/min/pmol of rhodopsin]. Rhodopsin, isolated in the presence of 1 per cent digitonin following ROS solubilization with 1 per cent digitonin, was capable of stimulating slightly transducin GTPase activity, with an initial rate of 1 pmol of GTP hydrolyzed/min/pmol of rhodopsin. However, rhodopsin purified in the presence of 0.2 per cent n-dodecyl-beta-D-maltoside (DM), following ROS solubilization with either 1 per cent CHAPS or 1 per cent DM, stimulated the enzymatic activity of transducin in a light-dependent manner, with an initial rate of 5 pmol of Pi released/min/pmol of rhodopsin. Addition of 0.075 per cent egg phosphatidylcholine (PC) to the four different solubilized rhodopsin samples significantly enhanced light-stimulated GTP hydrolysis by transducin, with initial rates increasing from 0 to 1, 1 to 2, and 5 to 30 pmol of Pi released/min/pmol of rhodopsin, respectively. Furthermore, DM-solubilized rhodopsin induced the hydrolysis of the maximun amount of GTP by transducin at 0.0075 per cent PC, while digitonin-solubilized rhodopsin only stimulated the GTPase activity of transducin to a similar value, when the amount of the photoreceptor protein was increased 4-fold and 0.15 per cent PC was added to the assay mixture. These results suggest that the effective photoactivation of transducin by rhodopsin requires phospholipids, which seem to be differentially eliminated with the detergent extraction procedure utilized during ROS membranes solubilization and photopigment isolation.

Improved purification of transducin subunits from bovine retinal rod outer segments

Transducin serves as a mediator between the receptor protein, rhodopsin, and the effector protein, cGMP phosphodiesterase, in the visual process. Transducin is a protein composed of three polypeptides: T alpha, T beta, and T gamma, and acts as two functional units, the alpha-subunit and the beta gamma-complex. In the present study, I describe an efficient and fast method of purifying T alpha and T beta gamma using chromatography on a blue agarose column connected in tandem with an omega-amino octylagarose column. The recombination of T alpha and T beta gamma reconstitutes the functional heterotrimeric holoprotein, as demonstrated by the recovery of three native properties of transducin: 1) its capacity to exchange guanine nucleotide, 2) its GTP hydrolytic activity, and 3) the ADP-ribosylation of T alpha catalysed by pertussis toxin