Cyclic nucleotide-dependent phosphorylation of proteins in rod outer segments in frog retina. Characteristics of the phosphorylated proteins and their dephosphorylation.
To clarify the function of cyclic nucleotides in rod outer segments (ROS) of frog retinas, we studied cyclic nucleotide-dependent phosphorylation and dephosphorylation of protein. cGMP or cAMP with [gamma-32P]ATP in the dark enhanced the phosphorylation of two ROS proteins with Mr = 10,500 (Band 1) and 8,500 (Band 2) according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The phosphorylation was maximally enhanced at 2.0 mM cGMP and cAMP in the presence of Mg2+. The cGMP-activated protein kinase showed near-optimal activity between pH 6.5 and 8.0. GMP, GDP, GTP, AMP, and ADP did not enhance the phosphorylation. The stoichiometry of the phosphate incorporated into Bands 1 and 2 could not be calculated because the amount of Bands 1 and 2 was too small to measure. Both 32P-phosphorylated Bands 1 and 2 (32P-Bands 1 and 2) were solubilized during preparation and the molecular weight of each, in the native preparation, was 19,000. Their isoelectric point was 5.2. The sites of phosphorylation were the serine residue(s). DEAE-Sephadex A-50 column chromatography gave a good separation of Bands 1 and 2 from other 32P-phosphoproteins at 60 mM NaCl. Dephosphorylation of 32P-Bands 1 and 2 in dark-adapted ROS suspension required Mn2+ or Mg2+; the former was more effective than the latter at concentrations below 0.5 mM. Both phosphorylation and dephosphorylation were inhibited by Zn2+.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001001:Anura; D002413:Cations, Divalent / Q000494:pharmacology; D002499:Centrifugation, Density Gradient; D002852:Chromatography, Ion Exchange; D000242:Cyclic AMP / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D010786:Photoreceptor Cells / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism*
D002413:Cations, Divalent; D010750:Phosphoproteins; D008345:Manganese; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D006152:Cyclic GMP; D008274:Magnesium