Phosphorylation in sealed rod outer segments: effects of cyclic nucleotides.
Rod outer segments (ROS) from rat were purified on Percoll gradients. These ROS had intact plasma membranes since they were impermeable to small molecules. Protein phosphorylation in the purified ROS was studied after the plasma membrane was disrupted by freeze/thawing. [gamma-32P]ATP was used as phosphate donor. ATP concentration, time, temperature, and light or dark adaptation were varied in the assays. The 32P-labeled proteins were separated by polyacrylamide gel electrophoresis and autoradiographed. Rhodopsin was the dominant phosphorylated protein, and the addition of adenosine cyclic 3',5'-phosphate (cAMP) or guanosine cyclic 3',5'-phosphate (cGMP) (10(-4) M) did not qualitatively alter the ROS phosphorylation pattern. The only cyclic nucleotide effect we could establish in these experiments was the inhibition of rhodopsin phosphorylation by cGMP. This inhibition did not appear to be competitive with ATP since cAMP was much less inhibitory than cGMP and the phosphorylation in the presence of cGMP reached a plateau at a much lower level than in control conditions. Hypotheses implying an involvement of protein phosphorylation/dephosphorylation in dark adaptation have been formulated [Miller, J. A., & Paulsen, R. (1975) J. Biol. Chem. 250, 4427-4432; Kuhn, H., McDowell, J. H., Leser, K. H., & Bader, S. (1977) Biophys. Struct. Mech. 3, 175-180]; we suggest that cGMP may control this process through the modulation of the extent of inhibition of phosphorylation of the visual pigment.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D000242:Cyclic AMP / Q000494:pharmacology*; D006152:Cyclic GMP / Q000494:pharmacology*; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000302:isolation & purification; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D010786:Photoreceptor Cells / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D012374:Rod Cell Outer Segment / Q000378:metabolism*
D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D006152:Cyclic GMP