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Rhodopsin is phosphorylated in a light-dependent manner by a kinase intrinsic to the rod outer segment. We have used chromatofocusing to separate six phosphorylated species of rhodopsin and have recovered in the pH gradient fractions 60-80% of the initial phosphorylated sample loaded on the column. The isolated species of rhodopsin coincide with the species that are observed in isoelectric focusing gels in the pH range 6.1-4.7. Unphosphorylated rhodopsin focuses at a pI of 6.0. Two species having two phosphates per rhodopsin with isoelectric points of 5.45 and 5.40 have been isolated. The phosphate to rhodopsin ratios for the remaining species are 3.8, 5.0, 6.1, and 8.2 with isoelectric points of 5.16, 4.99, 4.85, and 4.73, respectively. The chromatofocusing profile suggests that there may be multiple forms of rhodopsin with the same number of phosphates among some of the other phosphorylated forms of rhodopsin.

Recombinant membranes were prepared from phospholipid-free rhodopsin and egg phosphatidylcholine (PC) under a wide variety of conditions employing an octyl beta-D-glucoside (OG) dialysis procedure. Two bands were consistently observed after sucrose density centrifugation of these recombinants. The major band, which was protein rich, had a molar phospholipid:protein ratio that was in the range of 30:1 to 50:1, even when the molar phospholipid:protein ratio of the solubilized solution prior to OG removal was as high as 300:1. Similar results were obtained when dioleoyl-PC, 1-palmitoyl-2-oleoyl-PC, disk lipids, or diphytanoyl-PC was used instead of egg PC. These results can be explained in terms of a lower stability of the OG-phospholipid micelles relative to the OG-phospholipid-rhodopsin micelles. Of the phospholipids that were used in the OG dialysis procedure, only saturated dimyristoyl-PC produced a protein-rich recombinant band with a phospholipid:protein ratio close to that of the initial solubilized solution. In contrast to the results obtained by using OG, when solubilized disks supplemented with egg PC were reconstituted from sodium cholate or dodecyltrimethyl-ammonium bromide, the resulting recombinant membranes had initial and final phospholipid:protein ratios which were similar.

The solubilization of large, unilamellar egg phosphatidylcholine vesicles by the nonionic detergent octyl glucoside (OG) was investigated by nuclear magnetic resonance (NMR), fluorescence anisotropy, turbidity, electron microscopy, and centrifugation followed by compositional analysis. The solubilization process is well described by the three-stage model previously proposed for other detergents. In stage I, the OG partitions between the bilayer and aqueous phases with a molar partition coefficient of 59 +/- 6. The presence of OG in the bilayers produces a small "fluidizing" effect, as indicated by changes in the NMR and fluorescence anisotropy parameters. A rearrangement that forms large mixed bilayers occurs in the latter part of stage I. Stage II, the conversion of detergent-saturated bilayers into mixed micelles, begins at a ratio of total OG concentration minus the critical micelle concentration to total phosphatidylcholine concentration of approximately 1.5 and continues until this ratio reaches about 3.0. The correction for the critical micelle concentration of the OG is necessary for comparison of experimental results obtained at different lipid concentrations. The mixed bilayer-mixed micelle interconversion is quantified by the centrifugation experiments and by 31P NMR. The agreement between the two methods is excellent. Advantages of the NMR method are discussed. In stage III, which was not studied in detail here, all of the phosphatidylcholine is present as mixed micelles. Evidence is presented that the various structures present in the dispersions are in equilibrium with one another during these experiments.