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The reactivity of the sulfhydryl groups of rhodopsin in rod outer segment membranes
THE REACTIVITY OF THE SULFHYDRYL RHODOPSIN IN ROD OUTER SEGMENT J.
GROUPS OF MEMBRANES
HUGH MCDOWELL, MARIA T. MAS, KEAN D. GRIFFITH
and PAUL A. HARGRAVE School of Medicine and the Depa~ment of Chemistry and Bj~hemistry, Southern Illinois University, Carbondaie, Illinois 62901. USA. (Rrwiued
26 Ocrobcr
1978)
Abstract-The number of light-exposed sulfhydryl groups observed in rod outer segment suspensions is greatly influenced by the specific reaction conditions used. If appropriate conditions are chosen, the same pattern of sulfhydryl group reactivity is observed in rod outer segment suspensions as previously reported for rhodopsin in digitonin solution. It is not clear whether the influence of the reaction conditions on the pattern of sullhydryl reactivity reflects differences in the environment of the sulfhydryl groups, differences in the oxidation reactions, or some combination of these effects.
Jn this ~ommuni~tion we present data which suggests that under appropriate conditions, the reactivity of the su~ydryl groups of rhodopsin in rod outer segment membrane suspensions is the same as that of rhodopsin solubilized in digitonin solutions. The reactivity of the sulfhydryl groups of rhodopsin in digitonin solution has been fairly well characterized (Wald and Brown, 1952; De Grip et aI., 1973; Kimble and Ostroy, 1973; Zorn, 1974; De Grip et al., 1975; McDowell and Williams, 1976). Two sulthydryl groups can be modified in the dark by a variety of reagents, and an additional two become exposed upon illumination. Several similar studies in rod outer segments (ROS)’ have been reported (De Grip et al., 1973; Robertson et al., 1974; De Grip rt al., 1975; Chen and Hubbell, 1978). There are apparent differences in the observed reactivities of sulfhydryl groups in digitonin solution as compared to ROS suspensions, particularly with regard to the light-induced reactions. However, some ‘indication that these reactivities are in fact similar has been observed (McDowell, 1974), and therefore a further examination of the reactivity of the sulfhydryl groups in ROS suspensions was undertaken. Table 1 shows the reactivity of the sulfhydryl groups of ROS in the dark (see McDowell and Kuhn (1977) for preparation of ROS and determination of rhodopsin con~ntration~ Our values of 2.85 DTNBreactive sul~ydryls approach the 2-2.3 found by other workers, if our data are also corrected for the contribution of bleached rhodopsin (Chen and Hubbell, 1978). “C-NEM reacts with -2 sulthydryl groups as determined either by a filter assay of the ROS suspension or by chromatographically purifying the labeled rhodopsin. Prior reaction of ROS with NEM almost completely blocks the reaction of DTNB with the outer segments indicating that DTNB and NEM react with the same two sulfhydryl r Abbreviations used: ROS = rod outer segments; DTNB = 5.S-dithiobis-2~nitro~nzoic acid): NEM = Nethyl~tei~ide; IAS = l;~iodoa~tamido~~i~ylate; IAA = iodoacetic acid; HTP = hydroxyapatite. 1143
groups. On the other hand, IAA reacts with only one sul~ydryl group. IAS, a derivative of IAA, also reacts with only one sulfhydryl group per rhodopsin, leaving one sulfhydryl group to react with NEM. This suggests that IAS reacts with a single specific cysteine in the rhodopsin sequence which is distinct from the remaining NEM-reactive cysteine. This is in agreement with the finding of Sale et al., (1977) that the IAS-reactive site and the remaining NEM-reactive site are separate and distinct, and are located in different regions in the primary structure of rhodopsin. Data have been obtained in our laboratory that demonstrate the site specificity of the IAS reagent (McDowell and Griffith, 1978) and will be reported elsewhere. A variety of experimental conditions were evaluated in measuring the exposure of additional rhodopsin sulfhydryi groups following bleaching. Most consistent results were obtained when the dark-reactive sulthydryls were first irreversibly blocked with NEM. Results obtained at 35”37°C were more reproducible than those obtained at lower temperatures. As shown in Table 2, -2 sulfhydryl groups were reacted by bleaching in the presence of DTNB. These determinations were performed under constant illumination. Bleaching a sample in the presence of DTNB at 35°C for 5 min followed by an 85 mm dark incubation gave 0.97 f 0.03 (2) su~ydryls per rh~opsin as compared to I.85 t_ 0.02 (2) when incubated in continuous i&rmination. This is in substantial agreement with the data of Chen and Hubbell (1978). Using other reagents, NEM or IAA, approximately one lightexposed sulfhydryl group was detected. We concluded that at least two sulfhydryl groups may be exposed on bleaching ROS suspensions as has been previously observed in digitonin solution. Current investigations are underway to determine if IAA and/or NEM label specific light-exposed sites. The two conditions used in Table 2 that have not been used in previous studies of the reactivity of the ~~ydryl groups in ROS suspensions are the prior blocking of the dark-reactive sul~ydryl groups with NEM, and bleaching at 3537°C. When the “dark”
1144
J. HUGH MTDOWI:LLrt u/. Table 1. Reactivity of sulfhydryl groups of rod outer segment membranes In the dark Sample
Reagent
Conditions
1. ROS
DTNB
I hr dark 25°C. pH 8.0 6 hr dark
2.29 * o.052(9)
25‘C, pH 7.0 purify on HTP
2.06 + O.lY (8)
2. (a) ROS
14C-NEM
(b) ROSW.,W,~
Sulfhydryls per rhodopsin 2.85 + 0.181 (7)
column
3. ROS
‘%IAA
4. ROS,As5 5.
ROSN,.M3
“‘C-NEM
DTNB
7.5 hr dark 3O”C,pH 8.0
0.842(3)
6 hr dark, 25°C
0.89 f 0.014 (3)
pH 7.0. purify on HTP column 1hr dark
0.23 + 0.07’ (4)
25°C. pH 8.0
’ Determined using Ellman’s (1959)procedure. * Determined as reagent bound per rhodopsin using a filter assay similar to that of McDowell and Kiihn (1977)used for determining ‘*PO, bound to rhodopsin. 3 Treat ROS with 40 fold excess NEM at pH 7.0, 25°C for 3-6 hr in the dark, then wash out excess NEM. 4 Determined as moles NEM/rhodopsin contained in a pooled fraction of the purified rhodopsin. ’ Treat ROS with 20 fold excess of IAS at pH 7.0, 25”C, for 8 hr in the dark (Sale et al., 1977), then wash out excess IAS.
sulthydryl groups were reacted with DTNB instead of NEM, or when bleaching was performed at lower temperatures, fewer light-exposed sulfhydryls were observed as determined with DTNB. There is some indication that bleaching leads to promotion of oxidation reactions. For example, if bleaching is performed in the presence of 5-thio-2-nitrobenzoic acid FNB, the indicator resulting from the reaction of DTNB with sulfhydryl groups) often a loss in the concentration of TNB has been observed. Similarly, if a NEM-treated ROS sample is bleached in the presence of DTNB and then the solution is made 1% in SDS, fewer sulfhydryl groups were observed than in a duplicate sample that was kept dark. In a final experiment, ROS were treated with DTNB and the excess DTNB was removed by washing (Table 2, sample 4). When this suspension was bleached, about one indicator was released per rhodopsin. Again, bleaching at 35°C was important since
bleaching at lower temperatures gave fewer indicators released. This result is virtually identical to the release observed upon bleaching DTNB-treated rhodopsin in digitonin solution (McDowell and Williams, 1976). In rod outer segment suspensions, the specific reaction conditions greatly influence the number of lightexposed sullhydryl groups observed for rhodopsin. We conclude that if appropriate reaction conditions are chosen, rhodopsin in rod outer segment suspension will show the same pattern of sullhydryl reactivity that has been previously reported for rhodopsin in digitonin solution. However, the reactions are more easily demonstrated and reproduced in the homogeneous detergent-solubilized system. The influence of reaction conditions on sulfhydryl reactivity could reflect differences in the environment of the sulfhydryls or it could affect the oxidation reactions, or some combination of the two. The present data does not allow us to distinguish between these mechanisms.
Table 2. Light-exposed sulfhydryl groups in rod outer segment suspension Sample 1. ROSNkM1
Reagent
Conditions
DTNB
bleach’ 1 hr
Sullhydryls per rhodopsin 1.97 + 0.1g3 (4)
35°C. pH 8.0 2. ROSNtM1
“‘C-NEM
3. ROSNEM1
‘%IAA
4. ROSDTNsS
none
bleach’ 6 hr 35-37”C, pH 7.0 bleach’ 2.5-S hr 37”C, pH 8.0 bleach’ 1 hr 35”C, pH 8.0
0.83 + 0XX4(4) 1.0 * o.24 (2) 0.87 + 0.196 (4)
’ See footnote 3 Table 1. ’ Samples about 25 cm from a 150 watt flood lamp. 3 See footnote 1 Table 1. 4 See footnote 2 Table 1. 5 Treat ROS with 40-fold excess DTNB at pH 8.0, 35°C for 30 min in dark, then wash out excess DTNB. 6 Indicators released on bleaching.
Reactivity of sulfhydryl groups of rhodopsin Acknow/~~d~er,lPnrs-We wish to thank Mr Dennis P. Smith for preparmg the IAS reagent. This work was supported by DHEW Fellowship EY 05114 (J.H.M.), NIH grant EY 01275 (P.A.H.), and National Science Foundation Grant No. SMI 76-83630 for Undergraduate Research Participation (K.D.G.).
1145
McDowell J. H. (1974) A disulfide exchange model for the bleaching and regeneration of rhodopsin. Dissertation, Florida State University, Tallahassee, Florida. McDowell J. H. and Kuhn H. (1977) Light-induced phosphorylation of rhodopsin in cattle photoreceptor membranes: Substrate activation and inactivation. Biochemisfry 16, 40544060.
REFERENCES
Chen Y. S. and Hubbell W. L. (1978) Reactions of the sulfhydryl groups of membrane-bound bovine rhodopsin. Mrnihra~~r Biochem. I, IO7- I 30. De Grip W. J.. Bonting S. L. and Daemen F. J. M. (1975) Biochemical aspects of the visual process. XXVIII. Classification of sullhydryl groups in rhodopsin and other photoreceptor membrane proteins. Biochim. hiophys. Acta 3%, lO+lI5. De Grip W. J.. van de Laar Cl. L. M.. Daemen F. J. M. and Bonting S. L. (1973) Sullhydryl groups and rhodopsin ohotolvsis. Biochin~. hionhw. Acta 325. 315-322. Ellman G. L. (1959) Tissue sulfhydryl groups. Archs Biothem.
Biophys.
82, 7&77.
Kimble E. A. and Ostroy S. E. (1973) Kinetics of reaction of the sulfhydryl groups of rhodopsin. Biochirn. hiophys. Actu 325. 323-33 I.
McDowell J. H. and Griffith K. (1978) Localization of a dark reactive cysteine residue of rhodopsin. Invest. Ophthal. and Msual Sci. (Suppl.) 17, 125. McDowell J. H. and Williams T. P. (1976) Oxidation states of four sulfurs of rhodopsin before and after bleaching. Vision Res. 16, 643-646. Robertson G. A., Bello A. G., Stevenson W. D. and Rockey J. H. (1974) Characterization of a photoexposed sulfhydryl group of bovine rhodopsin available for chemical modification. Biochem. hiophys. Res. Commun. 59, 1151-1156. Sale G. J., Towner P. and Akhtar M. (1977) Functional rhodopsin complex consisting of three noncovalently linked fragments. Biochem. J. 16, 564-5644. Wald G. and Brown P. K. (1952) The role of sulfhydryls in the bleaching and synthesis of rhodopsin. J. gen. Physiol. 35. 797-821. Zorn M. (1974) The effect of blocked sullhydryl groups on the regenerability of rhodopsin. Expl Eye Res. 19, 215-221.
GROUPS OF MEMBRANES
HUGH MCDOWELL, MARIA T. MAS, KEAN D. GRIFFITH
and PAUL A. HARGRAVE School of Medicine and the Depa~ment of Chemistry and Bj~hemistry, Southern Illinois University, Carbondaie, Illinois 62901. USA. (Rrwiued
26 Ocrobcr
1978)
Abstract-The number of light-exposed sulfhydryl groups observed in rod outer segment suspensions is greatly influenced by the specific reaction conditions used. If appropriate conditions are chosen, the same pattern of sulfhydryl group reactivity is observed in rod outer segment suspensions as previously reported for rhodopsin in digitonin solution. It is not clear whether the influence of the reaction conditions on the pattern of sullhydryl reactivity reflects differences in the environment of the sulfhydryl groups, differences in the oxidation reactions, or some combination of these effects.
Jn this ~ommuni~tion we present data which suggests that under appropriate conditions, the reactivity of the su~ydryl groups of rhodopsin in rod outer segment membrane suspensions is the same as that of rhodopsin solubilized in digitonin solutions. The reactivity of the sulfhydryl groups of rhodopsin in digitonin solution has been fairly well characterized (Wald and Brown, 1952; De Grip et aI., 1973; Kimble and Ostroy, 1973; Zorn, 1974; De Grip et al., 1975; McDowell and Williams, 1976). Two sulthydryl groups can be modified in the dark by a variety of reagents, and an additional two become exposed upon illumination. Several similar studies in rod outer segments (ROS)’ have been reported (De Grip et al., 1973; Robertson et al., 1974; De Grip rt al., 1975; Chen and Hubbell, 1978). There are apparent differences in the observed reactivities of sulfhydryl groups in digitonin solution as compared to ROS suspensions, particularly with regard to the light-induced reactions. However, some ‘indication that these reactivities are in fact similar has been observed (McDowell, 1974), and therefore a further examination of the reactivity of the sulfhydryl groups in ROS suspensions was undertaken. Table 1 shows the reactivity of the sulfhydryl groups of ROS in the dark (see McDowell and Kuhn (1977) for preparation of ROS and determination of rhodopsin con~ntration~ Our values of 2.85 DTNBreactive sul~ydryls approach the 2-2.3 found by other workers, if our data are also corrected for the contribution of bleached rhodopsin (Chen and Hubbell, 1978). “C-NEM reacts with -2 sulthydryl groups as determined either by a filter assay of the ROS suspension or by chromatographically purifying the labeled rhodopsin. Prior reaction of ROS with NEM almost completely blocks the reaction of DTNB with the outer segments indicating that DTNB and NEM react with the same two sulfhydryl r Abbreviations used: ROS = rod outer segments; DTNB = 5.S-dithiobis-2~nitro~nzoic acid): NEM = Nethyl~tei~ide; IAS = l;~iodoa~tamido~~i~ylate; IAA = iodoacetic acid; HTP = hydroxyapatite. 1143
groups. On the other hand, IAA reacts with only one sul~ydryl group. IAS, a derivative of IAA, also reacts with only one sulfhydryl group per rhodopsin, leaving one sulfhydryl group to react with NEM. This suggests that IAS reacts with a single specific cysteine in the rhodopsin sequence which is distinct from the remaining NEM-reactive cysteine. This is in agreement with the finding of Sale et al., (1977) that the IAS-reactive site and the remaining NEM-reactive site are separate and distinct, and are located in different regions in the primary structure of rhodopsin. Data have been obtained in our laboratory that demonstrate the site specificity of the IAS reagent (McDowell and Griffith, 1978) and will be reported elsewhere. A variety of experimental conditions were evaluated in measuring the exposure of additional rhodopsin sulfhydryi groups following bleaching. Most consistent results were obtained when the dark-reactive sulthydryls were first irreversibly blocked with NEM. Results obtained at 35”37°C were more reproducible than those obtained at lower temperatures. As shown in Table 2, -2 sulfhydryl groups were reacted by bleaching in the presence of DTNB. These determinations were performed under constant illumination. Bleaching a sample in the presence of DTNB at 35°C for 5 min followed by an 85 mm dark incubation gave 0.97 f 0.03 (2) su~ydryls per rh~opsin as compared to I.85 t_ 0.02 (2) when incubated in continuous i&rmination. This is in substantial agreement with the data of Chen and Hubbell (1978). Using other reagents, NEM or IAA, approximately one lightexposed sulfhydryl group was detected. We concluded that at least two sulfhydryl groups may be exposed on bleaching ROS suspensions as has been previously observed in digitonin solution. Current investigations are underway to determine if IAA and/or NEM label specific light-exposed sites. The two conditions used in Table 2 that have not been used in previous studies of the reactivity of the ~~ydryl groups in ROS suspensions are the prior blocking of the dark-reactive sul~ydryl groups with NEM, and bleaching at 3537°C. When the “dark”
1144
J. HUGH MTDOWI:LLrt u/. Table 1. Reactivity of sulfhydryl groups of rod outer segment membranes In the dark Sample
Reagent
Conditions
1. ROS
DTNB
I hr dark 25°C. pH 8.0 6 hr dark
2.29 * o.052(9)
25‘C, pH 7.0 purify on HTP
2.06 + O.lY (8)
2. (a) ROS
14C-NEM
(b) ROSW.,W,~
Sulfhydryls per rhodopsin 2.85 + 0.181 (7)
column
3. ROS
‘%IAA
4. ROS,As5 5.
ROSN,.M3
“‘C-NEM
DTNB
7.5 hr dark 3O”C,pH 8.0
0.842(3)
6 hr dark, 25°C
0.89 f 0.014 (3)
pH 7.0. purify on HTP column 1hr dark
0.23 + 0.07’ (4)
25°C. pH 8.0
’ Determined using Ellman’s (1959)procedure. * Determined as reagent bound per rhodopsin using a filter assay similar to that of McDowell and Kiihn (1977)used for determining ‘*PO, bound to rhodopsin. 3 Treat ROS with 40 fold excess NEM at pH 7.0, 25°C for 3-6 hr in the dark, then wash out excess NEM. 4 Determined as moles NEM/rhodopsin contained in a pooled fraction of the purified rhodopsin. ’ Treat ROS with 20 fold excess of IAS at pH 7.0, 25”C, for 8 hr in the dark (Sale et al., 1977), then wash out excess IAS.
sulthydryl groups were reacted with DTNB instead of NEM, or when bleaching was performed at lower temperatures, fewer light-exposed sulfhydryls were observed as determined with DTNB. There is some indication that bleaching leads to promotion of oxidation reactions. For example, if bleaching is performed in the presence of 5-thio-2-nitrobenzoic acid FNB, the indicator resulting from the reaction of DTNB with sulfhydryl groups) often a loss in the concentration of TNB has been observed. Similarly, if a NEM-treated ROS sample is bleached in the presence of DTNB and then the solution is made 1% in SDS, fewer sulfhydryl groups were observed than in a duplicate sample that was kept dark. In a final experiment, ROS were treated with DTNB and the excess DTNB was removed by washing (Table 2, sample 4). When this suspension was bleached, about one indicator was released per rhodopsin. Again, bleaching at 35°C was important since
bleaching at lower temperatures gave fewer indicators released. This result is virtually identical to the release observed upon bleaching DTNB-treated rhodopsin in digitonin solution (McDowell and Williams, 1976). In rod outer segment suspensions, the specific reaction conditions greatly influence the number of lightexposed sullhydryl groups observed for rhodopsin. We conclude that if appropriate reaction conditions are chosen, rhodopsin in rod outer segment suspension will show the same pattern of sullhydryl reactivity that has been previously reported for rhodopsin in digitonin solution. However, the reactions are more easily demonstrated and reproduced in the homogeneous detergent-solubilized system. The influence of reaction conditions on sulfhydryl reactivity could reflect differences in the environment of the sulfhydryls or it could affect the oxidation reactions, or some combination of the two. The present data does not allow us to distinguish between these mechanisms.
Table 2. Light-exposed sulfhydryl groups in rod outer segment suspension Sample 1. ROSNkM1
Reagent
Conditions
DTNB
bleach’ 1 hr
Sullhydryls per rhodopsin 1.97 + 0.1g3 (4)
35°C. pH 8.0 2. ROSNtM1
“‘C-NEM
3. ROSNEM1
‘%IAA
4. ROSDTNsS
none
bleach’ 6 hr 35-37”C, pH 7.0 bleach’ 2.5-S hr 37”C, pH 8.0 bleach’ 1 hr 35”C, pH 8.0
0.83 + 0XX4(4) 1.0 * o.24 (2) 0.87 + 0.196 (4)
’ See footnote 3 Table 1. ’ Samples about 25 cm from a 150 watt flood lamp. 3 See footnote 1 Table 1. 4 See footnote 2 Table 1. 5 Treat ROS with 40-fold excess DTNB at pH 8.0, 35°C for 30 min in dark, then wash out excess DTNB. 6 Indicators released on bleaching.
Reactivity of sulfhydryl groups of rhodopsin Acknow/~~d~er,lPnrs-We wish to thank Mr Dennis P. Smith for preparmg the IAS reagent. This work was supported by DHEW Fellowship EY 05114 (J.H.M.), NIH grant EY 01275 (P.A.H.), and National Science Foundation Grant No. SMI 76-83630 for Undergraduate Research Participation (K.D.G.).
1145
McDowell J. H. (1974) A disulfide exchange model for the bleaching and regeneration of rhodopsin. Dissertation, Florida State University, Tallahassee, Florida. McDowell J. H. and Kuhn H. (1977) Light-induced phosphorylation of rhodopsin in cattle photoreceptor membranes: Substrate activation and inactivation. Biochemisfry 16, 40544060.
REFERENCES
Chen Y. S. and Hubbell W. L. (1978) Reactions of the sulfhydryl groups of membrane-bound bovine rhodopsin. Mrnihra~~r Biochem. I, IO7- I 30. De Grip W. J.. Bonting S. L. and Daemen F. J. M. (1975) Biochemical aspects of the visual process. XXVIII. Classification of sullhydryl groups in rhodopsin and other photoreceptor membrane proteins. Biochim. hiophys. Acta 3%, lO+lI5. De Grip W. J.. van de Laar Cl. L. M.. Daemen F. J. M. and Bonting S. L. (1973) Sullhydryl groups and rhodopsin ohotolvsis. Biochin~. hionhw. Acta 325. 315-322. Ellman G. L. (1959) Tissue sulfhydryl groups. Archs Biothem.
Biophys.
82, 7&77.
Kimble E. A. and Ostroy S. E. (1973) Kinetics of reaction of the sulfhydryl groups of rhodopsin. Biochirn. hiophys. Actu 325. 323-33 I.
McDowell J. H. and Griffith K. (1978) Localization of a dark reactive cysteine residue of rhodopsin. Invest. Ophthal. and Msual Sci. (Suppl.) 17, 125. McDowell J. H. and Williams T. P. (1976) Oxidation states of four sulfurs of rhodopsin before and after bleaching. Vision Res. 16, 643-646. Robertson G. A., Bello A. G., Stevenson W. D. and Rockey J. H. (1974) Characterization of a photoexposed sulfhydryl group of bovine rhodopsin available for chemical modification. Biochem. hiophys. Res. Commun. 59, 1151-1156. Sale G. J., Towner P. and Akhtar M. (1977) Functional rhodopsin complex consisting of three noncovalently linked fragments. Biochem. J. 16, 564-5644. Wald G. and Brown P. K. (1952) The role of sulfhydryls in the bleaching and synthesis of rhodopsin. J. gen. Physiol. 35. 797-821. Zorn M. (1974) The effect of blocked sullhydryl groups on the regenerability of rhodopsin. Expl Eye Res. 19, 215-221.