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Light exposure of rhodopsin in rod outer segment (ROS) membranes activates several cyclic GMP phosphodiesterase (PDE) molecules via a GTP-binding protein (G protein). Both PDE and G protein are surface-associated (peripheral) enzymes, which may be extracted from ROS by hypotonic media, individually purified, and recombined in isotonic media with purified rhodopsin-phospholipid vesicles to yield membranes of low dark and high light phosphodiesterase activity. In isotonic media, the PDE strongly associates with phospholipid membranes as well as with ROS and rhodopsin-phospholipid membranes. Because only membrane-associated PDE is readily light activated, the PDE activity saturates when the available binding sites are occupied. At a constant G-protein concentration, the PDE activity observed at saturation is 4 times greater for unilamellar rhodopsin-phospholipid vesicles with a lipid to rhodopsin ratio of 460 than for those with a ratio of 120. Thus, PDE association with membrane in isotonic media is dependent on the phospholipid content rather than the rhodopsin content. Several G proteins per PDE are necessary to maximize the PDE activity of reconstituted membranes; therefore, a weak association between activated G protein and PDE is indicated. Both peripheral enzymes readily transfer between membrane surfaces. Rhodopsin-phospholipid vesicles devoid of enzyme activity were exposed to a light flash and then mixed in the dark in isotonic media with unilluminated ROS membranes which contained PDE and G protein. PDE activity was observed within 2 s after mixing. Subsequent separation and evaluation of the denser ROS membranes and the less dense vesicles demonstrated that both PDE and G protein were associated with the vesicles as well as the ROS membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

The successful reconstitution of rhodopsin, the rod outer segment (ROS) G protein, and the ROS phosphodiesterase (PDE) into partially polymerized bilayer membranes is described. Purified bovine rhodopsin (Rh) was inserted into performed partially polymerized lipid vesicles. Sonicated vesicles composed of approximately equal moles of dioleoylphosphatidylcholine (DOPC) (or 1-palmitoyl-2-oleoyl-phosphatidylcholine) and 1,2-bis(octadeca-2,4-dienoyl)phosphatidylcholine (DENPC) were photolyzed with 254-nm light to polymerize the DENPC and form domains of DOPC and polyDENPC in the vesicle wall. Rh-octyl glucoside (OG) micelles were slowly added to the vesicle suspension to give 15 mM OG (below the OG critical micelle concentration). The suspension was incubated and then dialyzed and purified on a sucrose gradient. Ultracentrifugation revealed a major Rh-lipid band which was harvested and found to contain a 100 +/- 10 phosphatidylcholine to rhodopsin ratio (Rh-polyDENPC/DOPC). The orientation of Rh in the membrane was determined by limited proteolytic digestion of Rh and by competitive inhibition of monoclonal antibody binding to solubilized disk membranes. Results were compared with control membranes of Rh-DOPC (1:43) prepared by insertion and Rh-phospholipid membranes prepared by detergent dialysis. Visual inspection of thermolysin proteolytic patterns of Rh indicates one major population cleaved at the carboxy terminus, as is found in disk membranes with an asymmetric arrangement of Rh. In contrast, proteolysis of a Rh-egg PC/PE (1:50/50) membrane (detergent dialysis) produced two Rh populations, which indicates a symmetric arrangement of Rh. The Rh-polyDENPC/DOPC (1:100) membranes were allowed to compete with solubilized, immobilized disk membranes for the monoclonal antibody R2-15 (specific for the amino-terminal region of Rh). They were intermediate between the asymmetric ROS disk membranes and the symmetric dialysis membranes in their ability to bind the R2-15 monoclonal antibody. The data indicate approximately 80% of the Rh's in Rh-polyDENPC/DOPC are in the normal orientation found in disks. These Rh-containing polymerized bilayer membranes demonstrated functionality as determined by chemical regeneration, kinetic spectrophotometry, and cGMP cascade reconstitution experiments. In the latter experiments the peripheral proteins, ROS G protein and PDE, bound with comparable efficiency to both the polymerized PC bilayers and egg PC bilayers. Thus the biocompatibility of the phosphatidylcholine membrane surface was maintained after polymerization of DENPC.

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