Amino acid sequence adjacent to a sulfhydryl group exposed on illumination of bovine rhodopsin.

Two sulfhydryl groups of bovine rhodopsin, available for chemical modification only after bleaching, were specifically labeled with radioactive iodoacetamide. The labeled protein was extensively reduced and alkylated and digested with pronase, and peptides were purified by gel filtration chromatography, anion and cation exchange chromatography, and high pressure liquid chromatography. Purified peptides were detected by their radioactivity, UV spectral properties, and by their fluorescence after reaction with fluorescamine. Sequence analysis of a highly purified peptide established the sequence S-carboxamido[14C]methyl cysteinyl-prolyl-glycine as the site of one of the light-exposed sulfhydryl groups of bovine rhodopsin.

Two sulfhydryl groups of bovine rhodopsin, available for chemical modification only after bleaching, were specifically labeled with radioactive iodoacetamide. The labeled protein was extensively reduced and alkylated and digested with pronase, and peptides were purified by gel filtration chromatography, anion and cation exchange chromatography, and high pressure liquid chromatography. Purified peptides were detected by their radioactivity, UV spectral properties, and by their fluorescence after reaction with fluorescamine. Sequence analysis of a highly purified peptide established the sequence S-carb~xamido['~C]methyl cysteinyl-prolyl-glycine as the site of one of the lightexposed sulfhydryl groups of bovine rhodopsin.
The amino acid residues of rhodopsin with altered reactivity or orientation after photobleaching include a chromophore binding site lysyl residue whose €-amino group is coupled through a protonated Schiff base linkage to retinal in the native visual pigment (1-3), seryl and/or threonyl residues which are phosphorylated upon photobleaching of rhodopsin in native membranes (4, 5), aromatic residues whose optical activity is altered on bleaching (6, 7), and two cysteinyl sulfhydryl groups which become available for chemical modification in detergent solutions of rod outer segment membranes only after bleaching (8-11). Considerable recent effort has been directed toward placing these reactive residues within the primary structure of bovine rhodopsin. The retinal binding site lysyl residue has been located in the smaller F2 fragment produced by digestion of rhodopsin in rod outer segment membranes with thermolysin (12). The F2 fragment also contains the sites of light-dependent phosphorylation, a dark-reactive cysteinyl group, and the COOH-terminal amino acid sequence (5,13,14). A dipeptide of sequence alanyl-(retinal) lysine, containing the chromophore binding site lysyl residue, recently has been isolated from pronase digests of reductively labeled bovine rhodopsin (15). The larger F1 fragment produced by thermolysin digestion of membrane-bound rhodopsin contains the NHs-terminal amino acid sequence, the sites of attachment of the carbohydrate residues, and four * This work was supported by United States Public Health Service Research Grants EY 01193, NS 13201, and Research Career Development Award NS 00201, and an unrestricted grant from Research to Prevent Blindness, Inc., New York. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. sulfhydryl groups, two of which are available for chemical modification only after bleaching (10,12,16). The sulfhydryl groups exposed on bleaching also have been located in a smaller fragment of apparent M, = 13,000 produced by chymotryptic cleavage of labeled bovine rhodopsin in rod outer segment membranes (10).
The present investigations establish the amino acid sequence of a tripeptide of bovine rhodopsin containing a lightexposed sulfhydryl group selectively labeled with radioactive iodoacetamide in detergent solution.

MATERIALS AND METHODS
Rod outer segments were isolated from dark-adapted frozen bovine retina (Hormel, MN) by isopycnic discontinuous sucrose density gradient ultracentrifugation. Rhodopsin was extracted from rod outer segments in 1.5% (w/v) cetyl(hexadecy1)trimethyl ammonium bromide (Eastman) in 5 mM EDTA, 50 mM Tris-HC1 buffer, pH 7.8, at 4 "C, and purified by Bio-Gel A-1.5m chromatography. The Asao/Aaoo ratios of the pooled fractions used in the present studies were 1.6-1.9. Rhodopsin and opsin concentrations were calculated using molar extinction coefficients, EXKI and EZHO, of 40,000 and 66,000, respectively.
Rhodopsin (5-8 X M) was first reacted with a 4-8-fold molar excess of nonradioactive iodoacetamide (recrystallized from ethanol and water) for 2 h at room temperature, after which a 4-8-fold molar excess of i~do['~C]acetamide (specific activity, 22.1 mCi/mmol, ICN) was added and the sample was bleached. The radioactive alkylation of SH groups was terminated by the addition of a large excess of nonradioactive iodoacetamide and the protein was passed over a column of Sephadex G-25 in 50% acetic acid. Labeled opsin was reduced with a 50-fold molar excess of dithiothreitol in 6 M guanidine-HCI, 2 mM EDTA, 0.5 M Tris-HC1 buffer, pH 8.5, under nitrogen at 30 "C for 16 h, and alkylated with a 55-fold molar excess of iodoacetic acid for 1 h. The reduced and alkylated protein was dialyzed at 4 "C against 5% acetic acid for 24 h and then against 30% acetic acid for 48 h and lyophylized. Alkylated opsin was digested with pronase (2-4% of opsin concentration; Streptomyces griseus protease, B Grade, Calbiochem) in 2% digitonin, 0.1 M Na-phosphate buffer, pH 7.2, at 33 "C (17, 18). Additional pronase was added every 6-8 h and the reaction was terminated after 32 h by dilution with distilled water and lyophylization.
Radiolabeled pronase peptides of opsin were isolated by sequential gel filtration in 22% formic acid on columns of Bio-Gel P-30, P-10, P-4, and P-2 (Bio-Rad). Peptides were further purified by HPLC' on a Cla reverse phase column (Waters Associates Model 660 solvent programmer). Anion exchange chromatography was performed on DAX-2 resin (Dionex) and cation exchange chromatography on Dowex 50 (AA-15) resin. Peptides in the ion exchange chromatography effluent were detected by reacting a small aliquot of each fraction with fluorescamine (Fluram, Roche) (11,19) in 0.1 M Na-phosphate buffer, pH 8.0, and determining the fluorescence in an Aminco-Bowman Ratio Recording spectrophotofluorometer (excitation, 392 nm; emission, 475 mn).
Labeled proteins and purified peptides were hydrolyzed at 110 "C  . Ten per cent of -each PTH fraction from the sequenator was taken for radioactivity determination.

RESULTS
Amino acid analysis and high voltage electrophoresis of hydrolyzed radiolabeled opsin demonstrated that 85 k 10% of the radioactivity co-eluted or co-migrated with added authentic S-carboxymethylcysteine (Fig. 1). The ratio of nonradioactive iodoacetamide (added to rhodopsin in the dark) to i~do['~C]acetamide (added immediately prior to bleaching) was either 1:l or 2:l. The specific activity of the i~do['~C] acetamide was corrected for dilution with nonradioactive io- min; pump B, 0 to 50% linear gradient of 10 mM KH2P04, pH 5.5, and 50% methanol. Flow rate, 1 ml/min, 1 min/fraction collected; detection system, UV absorption at 210 nm; sensitivity, 1 absorbance unit full scale; cpm/lO pl. Peak HP3 was further puritled for sequence analysis (Fig. 4). Further purification of peak HP2 did not yield a peptide suitable for sequence analysis. The sample was dissolved in 0.5 ml of 0.1% H3P04 and applied to a HPLC column (CI, reversed phase, attached to a guard column; temperature, 30 "C; pressure, 2200 psi). Pump A, 0.1% H3P04, 15 min; pump B, linear gradient (curve 6 on programmer), 0 to 100% of 60% CH&N and 40% of 0.1% HsP04 (g). Flow rate, 2 ml/min; 1 min/fraction. Chart speed, 0.5 cm/min. Detection system, UV absorption at 210 nm; sensitivity, 2.0 absorbance units full scale; cpm/lO pl. Peak HP3 obtained by HPLC subfractionation (Fig. 3) was applied to a low pressure anion exchange (DAX-2) column (column dimensions, 30 X 0.6 cm; temperature, 56 "C; flow rate, 11 ml/h, 20 min/fraction). The column was eluted first with 3% pyridine, pH 9.0, for 3.5 h and then with a continuous gradient (-. -) of 60 ml each of 3% pyridine, pH 9.0,0.5 N ovridine/acetate. DH 5.5, and 2.0 N pvridine/acetate. DH 5.0. A sample ii.5%) of each fraition was used for reaction with fliorescamine, and the radioactivity of 10 ,ul of each sample was measured. sulfhydryl groups exposed on bleaching rhodopsin was found to be 7.8 based on labeling experiments where the alkylation was performed as a function of pH.
Pronase digestion of extensively reduced and alkylated postbleaching-labeled (S-carboxamido['4C]methylcysteinyl) opsin produced a number of radioactive peptides separable by gel filtration, HPLC, and anion and cation exchange chromatography. The majority of these peptides, however, were found to be impure when subjected to amino acid and sequence analysis. A single highly purified radioactive peptide was isolated by sequential Bio-Gel P-30, P-4, and P-2 gel filtration chromatography followed by HPLC (Fig. 2), HPLC subfractionation (Fig. 3), and finally by anion exchange chromatography (Fig. 4). About 70% of the total radioactivity applied to the anion exchange column (14-17% of total radioactivity of the labeled opsin) was recovered as one peptide (Fig. 4). The peak fraction of this peptide (AE-46, Fig. 4) was used for sequence analysis.
Amino acid analysis of peptide AE-46 showed the presence of S-carboxymethylcysteine, proline, and glycine in molar ratios of 1.0:0.9:1.2, with a maximum contamination of 15% of other amino acids. The identity of the residues on sequence analysis was established by quantitation of recovered radioactivity, thin layer chromatography, HPLC, and amino acid analysis after back hydrolysis of the PTH derivatives. On analysis of PTH derivatives of this peptide, 97% of the recovered radioactivity was obtained in the first cycle. The sequence of peptide AE-46 was established to be S-carboxamido['4C]methylcysteinyl-prolyl-glycine, with yields of 97, 89, and 76%, respectively, for the 3 residues.

DISCUSSION
The identification of the sequence carboxamido['4C]methylcysteinyl-prolyl-glycine for the pronase peptide from labeled bovine rhodopsin establishes the location of one of the two sulfhydryl groups available for chemical modification only after bleaching. Specific labeling of the light-exposed sulfhy-dry1 groups of rhodopsin was accomplished by first treating the native visual pigment in the dark with nonradioactive iodoacetamide, and then illuminating the protein in the presence of iodo['4C]acetamide.
In separate experiments where rhodopsin was labeled with low concentrations of iodo['4C] acetamide alone, in the dark or after immediate or delayed bleaching, the ratio of light to dark incorporation of radiolabel was 1O:l and 2 mol of S-carboxamido['4C]methylcysteine were formed/m01 of bleached rhodopsin (9): The amino acid sequence of bovine rhodopsin has not yet been completely determined. Sequence analysis of 36 amino acid residues from the COOH terminus has placed 3 cysteinyl or half-cystyl residues at positions 26,27, and 33 (13,14). The cysteinyl residue at position 33 has been reported to have a dark-reactive sulfhydryl group (13). More recently, a fourth cysteinyl residue has been identified in the sequence of a cyanogen bromide fragment of bovine rhodopsin ( 15).3 The nature of this cysteinyl residue and its placement within the complete primary structure of bovine rhodopsin has not yet been fully defined. The sequence Cys-Pro-Gly containing the light-exposed sulfhydryl group differs from all of the reported partial sequences for bovine rhodopsin thus far available.
The physicochemical mechanisms leading to exposure for chemical modification of the previously unreactive sulthydryl groups of bovine rhodopsin, produced by photobleaching in rod outer segment membranes or detergent solution (9, lo), have yet to be fully understood.
Membrane-labeling and proteolytic cleavage experiments have shown that the light-exposed sulfhydryl groups alkylated with iodo['4C]acetamide are located on the NH*-terminal Fl fragment (10). The retinal binding site lysyl residue and the phosphorylation sites are located on the COOH-terminal F2 fragment (12). The Fl and F2 fragments may occupy separate domains in the native visual pigment molecule (23). The light-exposed sulfhydryl groups of the Fl domain are thus far the only newly reactive residues of this portion of the rhodopsin molecule produced by photobleaching, and therefore may reflect a conformational change in a molecular domain distant from the chromophore binding site which may be of importance in visual transduction. A full understanding of the role of the sulthydryl groups exposed upon bleaching awaits knowledge of their placement within the three-dimensional structure of the native and bleached rhodopsin molecules.
The postbleaching reactive sulfhydryl groups also furnish a useful focus for study of comparative amino acid sequences of rod and cone visual pigments from different species. The present work lays a foundation for such studies.