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On the existence of several isoelectric forms of bovine rhodopsin
Exp. Eye Res. (19’i6) 23, 281-284
On the Existence of Several Isoelectric Forms of Bovine Rhodopsin* JAMES J. PLANTSER AND EDWARD L. KEAX~-
The Lorand 8. Johnson Laboratory for Research,in Ophthalmology, Department of Surgery, Division of Ophthalmology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, U.S.A. focusing studies carried out on purified bovine rhodopsin revealed the presence of several forms which differed in their isoelectric points. Three major species of rhodopsin were detected having isoelectric points of 4.99, 5.33 and 5.91. Upon bleaching. only one form of opsin was generated which had an isoelectric point of 5.22.
Isoelectric
1. Introduction There is relatively little information available concerning the isoelectric point (PI) of rhodopsin. In 1940, Broda and Victor reported a value of 4.47 for frog visual purple. After bleaching, the p1 was 4.57. Radding and Wald (1956) obtained values of 5.2 to 5.57 for the p1 of bovine rhodopsin by means of acid-base titrations. Likewise, upon bleaching, the p1 became about 0.1 pH unit more alkaline. More recently, Huang, Molday a,nd Dreyer (1973) reported ~1’s of 6.0 and 5.6 for native and bleached bovine rhodopsin, respectively. The latter workers used the technique of polyacrylamide gel isoelectric focusing. Unlike these reports, we have found that purified rhodopsin comprises several species which have different ~1’s. Furthermore, upon photobleaching, a protein was produced that had a unique PI. These observations were made using the very sensitive, high resolution technique of isoelectric focusing on a natural pH gradient as described by Vesterberg and Svensson (1966).
2. Materials and Methods Rod outer segments (ROS) were isolated from frozen, dark adapted bovine retinas (Hormel and Co., Austin, Minn.) by the procedure of Papermaster and Dreyer (1974). Rhodopsin was extracted from ROS with 1% Emulphogene BC 720 (donated by GAP Corporation, New York) in 0.05 ivf-Tris-HCl buffer, pH 7.0, and purified further by chromatography on columns of calcium phosphate-Celite, followed by affinity chromatography on Concanavalin A-Sepharose (Pharmacia, Uppsala), as described by Plantner and Kean (1976). The A2,s/&s ratios of the rhodopsin preparations used in these studies va.ried from 1.8 to 2-O. The concentration of rhodopsin was calculated from the difference in absorbance measured at 498 nm, before and after photobleaching, using a molar extinction coefficient of 40 600 (Wald and Brown, 1953-4). Spectra and optical density readings were obtained with a Beckman model 25 double beam recording spectrophotometer. All operations were performed at 4°C unless indicated otherwise, and all steps involving native rhodopsin were carried out in dim red light (General Electric bulbs,
type BAS). * Reprint requests to Dr Edward L. &an. t Affiliated also with the Department of Biochemistry. 281
282
J. J. PLAXTNER
AKD
E. L. KEAN
Isoelectric focusing was performed on an LKB (Stockholm) model 8101 (110 ml) isoelectric focusing column using an LKB model 33710 power supply. Broad range (pH 3.5 to pH 10) and narrow range (pH 4 to pH 6) carrier ampholyte solutions were used (Ampholine carrier ampholytes, LKB) to prepare the pH gradients which were stabilized on a sucrose gradient (4-47%), as described by the manufacturer. In addition to the ampholytes and sucrose, Emulphogene BC 720 was present at a concentration of 0.3%. Solutions of purified rhodopsin (0.08-0.25 pmol) in 0.05 M-Tris-HCl buffer, pH 7.0, containing 0.3% Emulphogene, were applied near the middle of the sucrose gradient. The column was run at 400 V for 2 hr, then increased to 500 V for the duration of the run which was 5 days. The apparatus was operated in a cold room (4°C) and in addition, the column was maintained at this temperature by a circulating bath. At the completion of the run, 1.5 ml fractions were collected at a flow rate of 0.6 ml/min. The pH was measured immedia.tely, in the cold, using a model 320 expanded range pH meter (Fisher Scientific Co., Pittsburgh) and a Beckman (Palo Alto, Calif.) model 39183 combination electrode. Spectrophotometric readings were obtained using in the reference cuvette a mixture of equal volumes of dense and less-dense sucrose density solutions containing 1% of the appropriate carrier ampholyte.
3. Results Xative
,rhodopsin-multiple
forn~
In Fig. 1 is seen a typical pattern obtained when purified rhodopsin was subjected to electrofocusing. Three major forms were detected. Each contained A,,, and Aa9s absorbing material, and in addition, each displayed the typical spectrum of rhodopsin before and after photobleaching. Although there was variation in the absolute values of the ~1’s between different preparations of rhodopsin examined over a period of more than a year, the presence of the three major isoelectric forms was consistently
O-20 0 18 0. I6
Volume FIG.
affinity (pH4-6).
(ml)
1. Isoelectric focusing of bovine rhodopsin. Rhodopsin, 0.13 pmol. purified by adsorption and chromatography, was electrofocused as described in Methods using a narrow pH range gradient
pH C-J; 82,s Cm);&a (XL
observed. A summary of the isoelectric points of these several forms from different experiments is given in Table I. The p1 is taken as the pH at the peak of the curves designated A, B and C. The mean value for the p1 of the individual forms from 14 runs made on the six separate preparations of rhodopsin is essentially the same as that given in Table I showing the mean value based on the number of preparations.
ISOELECTRIC
FORMS
283
OF RHODOPSIX
TABLE
I
Isoelectric fornas of bovine rhodopsin Form*
Isoelectric
A B
point?
4.9910.12
(6)
C
533&0.11 5.91&0.09
(6) (6)
Opsint
5.22+0.13
(4)
* A, B and C refer to the pattern obtained from the focusing column as in Fig. 1. P The data is presented in the order: mean &s.E.M. (number of preparations). f See Fig. 2.
The three forms were present in approximately equal proportions under conditions in which over 8004, recovery was obtained of the A,,, absorbing material placed onto the column. This same focusing pattern was reformed when the fractions containing A; B and C were pooled, dialyzed, concentrated and then refocused. After bleachinp When purified rhodopsin was photobleached by exposure to room fluorescent light and then electrofocused, the pattern of multiple forms was lost. Instead a major .A’aie absorbing peak was obtained which had an isoelectric point of 5.22 (Fig. 2: Table I). The small peak of A2,s, A,, absorbing material which migrated more toward the cathode in Fig. 2 was not present in all runs. In addition, the pH a’t which 1.6
14
1.4
13
I.2
12
I.0 8 6 +2 0.8
IO
4
8
0.6
6
0.4
4
0.2
2
030
40
50
60
70
80
=L1
90”
Volume (ml) FIG. 2. Isoelectric focusing of photobleached rhodopsin. Purified bovine rhodopsin (0.09 pmol) was photobleached and subjected to isoelectric focusing as described in Methods, using broad pH range carrier Ampholytes. The recoverjr of the AzTs absorbing material applied to the column was 108%. PH (o-0): &s (A---A); &m (m ---m).
284
J. J. PLAXTNER
AND
E. L. KEAX
absorbing material emerged from the column varied according to the site the 4,s of application of the sample to the focusing column, suggesting that this material is not charged (retinal?). 4. Discussion
Purified rhodopsin was shown by these studies to contain species which differed from each other in terms of their isoelectric points. Three major forms were detected with PI’S of about 5.0, 5.3 and 5.9. The technique that was used here, although more time consuming than the polyacrylamide gel variety of isoelectric focusing, has the advantages of higher capacity and greater resolution. Preliminary observations using focusing columns of greater capacity suggest that it may be possible to detect subclasses within each of these three major groups (Plantner and Kean, unpublished observations). The basis for this plurality of isoelectric forms in purified visual pigment is not presently clear, and could reflect the influence of several factors such as the presence of phosphorylated rhodopsins (Bownds, Dawes, Miller and Stahlman, 1972), and the ionization states of the thermal intermediates of rhodopsin (Ostroy, 1974). As seen here, bleaching had a sufficiently extensive effect on the net charge on the protein such that opsin displayed a single p1 although derived from the multiple forms present in the native state. Exposure of sulfhydryl groups on the protein has been associated with the conformational changes resulting from the bleaching of rhodopsin (Wald and Brown, 1951-a), and may have contributed to these effects on the isoelectric point. Further studies are in process to characterize further the mukiple isoelectric forms of bovine rhodopsin. BCKNOWLEDGMEKTS
The excellent technical assistance of Mrs Chu C. Popiela is acknowledged. This work was supported by Public Health Service Research Grant EY 00393 to E. L. Kean, the Ohio Lions Research Foundation, and by Postdoctoral Fellowship Grant EY 03300 to J. J. Plantner. REFERENCES Bownds, D., Dawes, J., Mililrer, J. and Stahlman, IM. (1972). Phosphorylation of frog photoreceptor membranes induced by light. Nature, New Biol. 237, 125-7. Broda, E. E. and Victor, E. (1940). The cataphoretic mobility of visual purple. Biochem. J. 34, 1501-6. Huang, H. V., Molday, R. S. and Dreyer, W. J. (1973). Isoelectric focusing of rod outer segment membrane proteins. F.E.B.S. Lett. 37,28690. Ostroy, S. E. (1974). Hydrogen ion changes of rhodopsin. Arch. Bioehem. Biophys. 164, 275-84. Papermaster, D. S. and Dreyer, W. J. (1974). Rhodopsin content in the outer segment membranes of bovine and frog retinal rods. Biochemisky 13, 243544. Plantner, J. J. and Kean, E. L. (1976). Carbohydrate composition of bovine rhodopsin, J. Biol. Chem. 251,1548-52. Radding, C. RI. and Wald, G. (1956). The stability of rhodopsin and opsin. Effects of pH and aging. J. Gen. Physiol. 39, 923-33. Vesterberg, 0. and Svennson, H. (1966). Isoelectric fractionation, analysis and characterization of ampholytes in natural pH gradients. Acta Chem. Scud 20, 820-34. Wald, G. and Brown, P. K. (1951-2). The role of sulfhydryl groups in the bleaching and synthesis of rhodopsin. J. Gen. Physiol. 35,797X321. Wald, 0. and BroTvn, P. K. (1953-4). The molar extinction of rhodopsin. J. Gevz. Physiol. 37, 189-200.
On the Existence of Several Isoelectric Forms of Bovine Rhodopsin* JAMES J. PLANTSER AND EDWARD L. KEAX~-
The Lorand 8. Johnson Laboratory for Research,in Ophthalmology, Department of Surgery, Division of Ophthalmology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, U.S.A. focusing studies carried out on purified bovine rhodopsin revealed the presence of several forms which differed in their isoelectric points. Three major species of rhodopsin were detected having isoelectric points of 4.99, 5.33 and 5.91. Upon bleaching. only one form of opsin was generated which had an isoelectric point of 5.22.
Isoelectric
1. Introduction There is relatively little information available concerning the isoelectric point (PI) of rhodopsin. In 1940, Broda and Victor reported a value of 4.47 for frog visual purple. After bleaching, the p1 was 4.57. Radding and Wald (1956) obtained values of 5.2 to 5.57 for the p1 of bovine rhodopsin by means of acid-base titrations. Likewise, upon bleaching, the p1 became about 0.1 pH unit more alkaline. More recently, Huang, Molday a,nd Dreyer (1973) reported ~1’s of 6.0 and 5.6 for native and bleached bovine rhodopsin, respectively. The latter workers used the technique of polyacrylamide gel isoelectric focusing. Unlike these reports, we have found that purified rhodopsin comprises several species which have different ~1’s. Furthermore, upon photobleaching, a protein was produced that had a unique PI. These observations were made using the very sensitive, high resolution technique of isoelectric focusing on a natural pH gradient as described by Vesterberg and Svensson (1966).
2. Materials and Methods Rod outer segments (ROS) were isolated from frozen, dark adapted bovine retinas (Hormel and Co., Austin, Minn.) by the procedure of Papermaster and Dreyer (1974). Rhodopsin was extracted from ROS with 1% Emulphogene BC 720 (donated by GAP Corporation, New York) in 0.05 ivf-Tris-HCl buffer, pH 7.0, and purified further by chromatography on columns of calcium phosphate-Celite, followed by affinity chromatography on Concanavalin A-Sepharose (Pharmacia, Uppsala), as described by Plantner and Kean (1976). The A2,s/&s ratios of the rhodopsin preparations used in these studies va.ried from 1.8 to 2-O. The concentration of rhodopsin was calculated from the difference in absorbance measured at 498 nm, before and after photobleaching, using a molar extinction coefficient of 40 600 (Wald and Brown, 1953-4). Spectra and optical density readings were obtained with a Beckman model 25 double beam recording spectrophotometer. All operations were performed at 4°C unless indicated otherwise, and all steps involving native rhodopsin were carried out in dim red light (General Electric bulbs,
type BAS). * Reprint requests to Dr Edward L. &an. t Affiliated also with the Department of Biochemistry. 281
282
J. J. PLAXTNER
AKD
E. L. KEAN
Isoelectric focusing was performed on an LKB (Stockholm) model 8101 (110 ml) isoelectric focusing column using an LKB model 33710 power supply. Broad range (pH 3.5 to pH 10) and narrow range (pH 4 to pH 6) carrier ampholyte solutions were used (Ampholine carrier ampholytes, LKB) to prepare the pH gradients which were stabilized on a sucrose gradient (4-47%), as described by the manufacturer. In addition to the ampholytes and sucrose, Emulphogene BC 720 was present at a concentration of 0.3%. Solutions of purified rhodopsin (0.08-0.25 pmol) in 0.05 M-Tris-HCl buffer, pH 7.0, containing 0.3% Emulphogene, were applied near the middle of the sucrose gradient. The column was run at 400 V for 2 hr, then increased to 500 V for the duration of the run which was 5 days. The apparatus was operated in a cold room (4°C) and in addition, the column was maintained at this temperature by a circulating bath. At the completion of the run, 1.5 ml fractions were collected at a flow rate of 0.6 ml/min. The pH was measured immedia.tely, in the cold, using a model 320 expanded range pH meter (Fisher Scientific Co., Pittsburgh) and a Beckman (Palo Alto, Calif.) model 39183 combination electrode. Spectrophotometric readings were obtained using in the reference cuvette a mixture of equal volumes of dense and less-dense sucrose density solutions containing 1% of the appropriate carrier ampholyte.
3. Results Xative
,rhodopsin-multiple
forn~
In Fig. 1 is seen a typical pattern obtained when purified rhodopsin was subjected to electrofocusing. Three major forms were detected. Each contained A,,, and Aa9s absorbing material, and in addition, each displayed the typical spectrum of rhodopsin before and after photobleaching. Although there was variation in the absolute values of the ~1’s between different preparations of rhodopsin examined over a period of more than a year, the presence of the three major isoelectric forms was consistently
O-20 0 18 0. I6
Volume FIG.
affinity (pH4-6).
(ml)
1. Isoelectric focusing of bovine rhodopsin. Rhodopsin, 0.13 pmol. purified by adsorption and chromatography, was electrofocused as described in Methods using a narrow pH range gradient
pH C-J; 82,s Cm);&a (XL
observed. A summary of the isoelectric points of these several forms from different experiments is given in Table I. The p1 is taken as the pH at the peak of the curves designated A, B and C. The mean value for the p1 of the individual forms from 14 runs made on the six separate preparations of rhodopsin is essentially the same as that given in Table I showing the mean value based on the number of preparations.
ISOELECTRIC
FORMS
283
OF RHODOPSIX
TABLE
I
Isoelectric fornas of bovine rhodopsin Form*
Isoelectric
A B
point?
4.9910.12
(6)
C
533&0.11 5.91&0.09
(6) (6)
Opsint
5.22+0.13
(4)
* A, B and C refer to the pattern obtained from the focusing column as in Fig. 1. P The data is presented in the order: mean &s.E.M. (number of preparations). f See Fig. 2.
The three forms were present in approximately equal proportions under conditions in which over 8004, recovery was obtained of the A,,, absorbing material placed onto the column. This same focusing pattern was reformed when the fractions containing A; B and C were pooled, dialyzed, concentrated and then refocused. After bleachinp When purified rhodopsin was photobleached by exposure to room fluorescent light and then electrofocused, the pattern of multiple forms was lost. Instead a major .A’aie absorbing peak was obtained which had an isoelectric point of 5.22 (Fig. 2: Table I). The small peak of A2,s, A,, absorbing material which migrated more toward the cathode in Fig. 2 was not present in all runs. In addition, the pH a’t which 1.6
14
1.4
13
I.2
12
I.0 8 6 +2 0.8
IO
4
8
0.6
6
0.4
4
0.2
2
030
40
50
60
70
80
=L1
90”
Volume (ml) FIG. 2. Isoelectric focusing of photobleached rhodopsin. Purified bovine rhodopsin (0.09 pmol) was photobleached and subjected to isoelectric focusing as described in Methods, using broad pH range carrier Ampholytes. The recoverjr of the AzTs absorbing material applied to the column was 108%. PH (o-0): &s (A---A); &m (m ---m).
284
J. J. PLAXTNER
AND
E. L. KEAX
absorbing material emerged from the column varied according to the site the 4,s of application of the sample to the focusing column, suggesting that this material is not charged (retinal?). 4. Discussion
Purified rhodopsin was shown by these studies to contain species which differed from each other in terms of their isoelectric points. Three major forms were detected with PI’S of about 5.0, 5.3 and 5.9. The technique that was used here, although more time consuming than the polyacrylamide gel variety of isoelectric focusing, has the advantages of higher capacity and greater resolution. Preliminary observations using focusing columns of greater capacity suggest that it may be possible to detect subclasses within each of these three major groups (Plantner and Kean, unpublished observations). The basis for this plurality of isoelectric forms in purified visual pigment is not presently clear, and could reflect the influence of several factors such as the presence of phosphorylated rhodopsins (Bownds, Dawes, Miller and Stahlman, 1972), and the ionization states of the thermal intermediates of rhodopsin (Ostroy, 1974). As seen here, bleaching had a sufficiently extensive effect on the net charge on the protein such that opsin displayed a single p1 although derived from the multiple forms present in the native state. Exposure of sulfhydryl groups on the protein has been associated with the conformational changes resulting from the bleaching of rhodopsin (Wald and Brown, 1951-a), and may have contributed to these effects on the isoelectric point. Further studies are in process to characterize further the mukiple isoelectric forms of bovine rhodopsin. BCKNOWLEDGMEKTS
The excellent technical assistance of Mrs Chu C. Popiela is acknowledged. This work was supported by Public Health Service Research Grant EY 00393 to E. L. Kean, the Ohio Lions Research Foundation, and by Postdoctoral Fellowship Grant EY 03300 to J. J. Plantner. REFERENCES Bownds, D., Dawes, J., Mililrer, J. and Stahlman, IM. (1972). Phosphorylation of frog photoreceptor membranes induced by light. Nature, New Biol. 237, 125-7. Broda, E. E. and Victor, E. (1940). The cataphoretic mobility of visual purple. Biochem. J. 34, 1501-6. Huang, H. V., Molday, R. S. and Dreyer, W. J. (1973). Isoelectric focusing of rod outer segment membrane proteins. F.E.B.S. Lett. 37,28690. Ostroy, S. E. (1974). Hydrogen ion changes of rhodopsin. Arch. Bioehem. Biophys. 164, 275-84. Papermaster, D. S. and Dreyer, W. J. (1974). Rhodopsin content in the outer segment membranes of bovine and frog retinal rods. Biochemisky 13, 243544. Plantner, J. J. and Kean, E. L. (1976). Carbohydrate composition of bovine rhodopsin, J. Biol. Chem. 251,1548-52. Radding, C. RI. and Wald, G. (1956). The stability of rhodopsin and opsin. Effects of pH and aging. J. Gen. Physiol. 39, 923-33. Vesterberg, 0. and Svennson, H. (1966). Isoelectric fractionation, analysis and characterization of ampholytes in natural pH gradients. Acta Chem. Scud 20, 820-34. Wald, G. and Brown, P. K. (1951-2). The role of sulfhydryl groups in the bleaching and synthesis of rhodopsin. J. Gen. Physiol. 35,797X321. Wald, 0. and BroTvn, P. K. (1953-4). The molar extinction of rhodopsin. J. Gevz. Physiol. 37, 189-200.