Molecular organization of bovine rod cGMP-phosphodiesterase 6.

Phosphodiesterase 6 (PDE6), a multisubunit (alphabetagamma(2)delta) enzyme, plays a major role in visual function by hydrolysing cGMP in response to a light stimulus. Solubilized bovine rod PDE6 molecules depleted of their gamma subunits were purified to homogeneity from bovine retinal rods and their molecular organization was investigated by electron microscopy. Image analysis of single particles revealed the three-dimensional dimeric arrangement of the purified alphabetadelta complex, and the internal organization of each catalytic subunit into three distinct domains at a resolution of 2.

Mono ADP-ribosylation of transducin catalyzed by rod outer segment extract.

Transducin is the retinal rod outer segment (ROS)-specific G protein coupling the photoexcited rhodopsin to cyclic GMP-phosphodiesterase. The alpha subunit of transducin is known to be ADP-ribosylated by bacterial toxins. We investigated the possibility that transducin is modified in vitro by an endogenous ADP-ribosyltransferase activity.

Conformational changes of cytosolic loops of bovine rhodopsin during the transition to metarhodopsin-II: an investigation by Fourier transform infrared difference spectroscopy.

In order to assign the structural changes of the protein, observed in the Fourier transform infrared (FT-IR) difference spectra of the rhodopsin-metarhodopsin-II transition, to specific regions of the protein, rhodopsin was treated by proteases. Nonilluminated and bleached rhodopsin was treated with protease K and papain. Rhodopsin digested in the bleached state was subsequently regenerated with 11-cis-retinal.

Interplay between calcium and activated cGMP phosphodiesterase from retinal rod outer segments.

Hypotonic extraction of bovine retinal rod outer segments after bleaching in isotonic buffer yielded an extract exhibiting activated cGMP phosphodiesterase properties. Since this extract was virtually devoid of other proteins involved in the rod outer segment cGMP enzymatic cascade, it was used to study phosphodiesterase catalytic activity. The hypotonic extract required Mg2+ in the range 0.