Using the radionuclide 65Zn, we have demonstrated the direct binding of zinc to purified rhodopsin. 65Zn is eluted with detergent-solubilized rhodopsin from concanavalin A columns and remains bound to the visual pigment through a subsequent gel-filtration step. Zinc binding to purified disc membranes is highly specific and, of the ions tested, copper is the best competitor. Equilibrium-dialysis experiments indicate that zinc binding to detergent-solubilized forms of rhodopsin may increase on bleaching the photopigment. These results may have important implications for studies that indicate that zinc plays a role in retinal degeneration and normal photoreceptor physiology.

Zinc can increase initial rhodopsin phosphorylation, apparently acting at the substrate rhodopsin and not at relevant phosphatases or rhodopsin kinase. How zinc binding to rhodopsin might increase its ability to serve as a substrate for phosphorylation is under investigation.

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.

Intact synaptosomes isolated from mammalian brain tissues (rat, mouse, gerbil, and human) have an ATP hydrolyzing enzyme activity on their external surface. The synaptosomal ecto‐ATPase(s) possesses characteristics consistent with those that have been described for ecto‐ATPases of various other cell types. The enzyme has a high affinity for ATP (the apparent Km values are in the range of 2–5 × 10−5M), and is apparently stimulated equally well by either Mg2+ or Ca2+ in the absence of any other cations. The apparent activation constant for both divalent cations is approximately 4 × 10−4M in all mammalian brain tissues studied. The involvement of a nonspecific phosphatase in the hydrolysis of externally added ATP is excluded. ATP hydrolysis is maximal in the pH range 7.4–7.8 for both divalent cation‐dependent ATPase activities. Dicyclohexylcarbodiimide, 2,4‐dinitrophenol, trifluoperazine, chlorpromazine, and p‐chloromercuribenzoate (50 μM) inhibit the ecto‐ATPase, whereas ouabain (1 mM) and oligomycin (3.5 μg × mg−1 protein) show little or no inhibition of this enzyme activity. Inhibitor data suggest that the Mg2+‐ and Ca2+‐dependent ecto‐ATPase may represent two different enzymes on the surface of synaptosomes.

8‐Azidoadenosine triphosphate labeled in the α or γ position with 32P was used as a photoaffinity reagent for identifying ATP binding sites on the external surface of intact rat brain synaptosomes. As revealed by autoradiography of sodium dodecyl sulfate‐polyacrylamide gel electrophoretic patterns, UV irradiation of intact synaptosomes in the presence of the above radioactive compounds at 5–10 µM resulted in the formation of several major radioactive conjugates with approximate molecular masses of 29, 45/46, 58, and 93 kDa. Minor bands of 20, 39, 52/54, 82/84, 120, and 140 kDa were also consistently labeled in these experiments. The possibility that labeling of these proteins was due to the presence of contaminating subcellular particles or intrasynaptosomal proteins was excluded. The major 8‐azidoadenosine [α‐32P]triphosphate‐labeled protein complex of ∼45/46 kDa was resolved into several subbands that are labeled differently depending on the type of divalent cations added to the photoaffinity reaction. In the presence of magnesium only, the major labeled band appeared at 45 kDa. With calcium, two additional subbands (43 and 46 kDa) could be distinguished. In the presence of 1 mM EDTA, a band at 44 kDa was labeled within this ATP‐binding complex. The labeling pattern of the subbands of this 45/46‐kDa complex is consistent with these bands being extracellular ATP‐binding proteins on the surface of the synaptosome.