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The green rod pigment of the bullfrog
RESEARCH NOTE THE GREEN ROD PIGMENT
OF THE BULLFROG
J. SILLsUN’
.&SOLD
Department of Biology. University of Pittsburgh, Pittsburgh. PA 15260, U.S.A. lRrceicen 9 July 1975)
In a study of rapid dark adaptation Sillman (1974) reported that the scotopic spectral sensitivity curve derived from the photoreceptors of the isolated. perfused bullfrog retina was much broader than that which should have been expected if only rhodopsin were involved. It was suggested that the elevation at the longer wavelengths mi_$tt be due, on the one hand, to cones which are acttve under scotopic conditions and, on the other hand, to the possible presence of porphyropsin (VP524,) a visual pinent which absorbs at longer wavelengths than bullfrog rhodopsin (VP504i) and which is sometimes present in the retina of the bullfrog (Reuter, White and Wald, 1971: Sillman, Owen and Femandez, 1972). It was also suggested that the elevation at the shorter wavelengths might be due, at least in part, to the presence of the green rod pigment which has been observed in the retinas of several other ranids and which absorbs maximally in the region of 435 nm (Denton and Wyllie, 1955: Dartnall, 1957, 1967; Donner and Reuter, 1962: Reuter. 1966: Liebman and Entine. 1968. See Crescitelli, 1972, for a review.). Since green rods have been described in the bullfrog retina (Steinberg, 1973), it certainly is a logical assumption that this retina contains the blue absorbing pigment typical of those photoreceptors. Surprisingly. however. there is no report in the literature of the presence or absence of such a visual pigment in the bullfrog retina, even though the bullfrog is a very common experimental animal in studies concerned with the visual system. The present work, therefore, was carried out to determine whether or not a blue absorbing photopigment does indeed exist in the retina of the bullfrog and, if so. what the properties are. Accordingly, an extract (2”; digitonin in 0.1 M phosphate buffer, pH 7.4) of bullfrog (Rana cnresbeiana) outer segments was subjected to a simple, two-step, partial bleaching analysis using the method of Dartnall (1952). Thus, the sample was exposed first to 600 nm light for 17 hr 35 min. a treatment which is expected to bleach nearly all visual pigment absorbing maximally at 500 nm or higher but which should leave untouched any visual pi_ment absorbing maximally in the blue region of the spectrum. After the lengthy exposure to 600nm light the sample was then exposed for 2 hr to light of 470nm, a wavelength which is expected to bleach any visual pigment absorbing in the blue. The results, displayed as difference spectra, are illustrated in Fig. 1. The filled circles (B) represent the change resulting from the exposure to 600nm light. ’ Present address: Dept. of Animal Physiology. University of California, Davis, CA 95616, U.S.A. 421
It is apparent that this red light bleached out pigment absorbing maximally near 5OOnm. In contrast, the subsequent exposure of the sample to 470nm light resulted in a decrease in optical density (A) which was maximal not in the region of 5OOnm, but close to 45Onm. From this it may be concluded that the extract contained a pigment absorbing in the blue region of the spectrum which is probably the visual pigment contained within the green rods. However, for two reasons the filled squares of Fig. 1 cannot accurately reflect the true spectral absorbance curve of the bullfrog’s green rod pigment. First, it is not unlikely that some small amount of rhodopsin remained in the sample even after the lengthy exposure to 600 nm light. This residual rhodopsin (VP504i) would be destroyed together with the green rod pigment upon exposure of the sample to 470nm light and, therefore, would tend to shift the resulting difference curve toward longer wavelengths. Second, and much more important, since the analysis was performed in the absence of hydroxylamine, product interference undoubtedly resulted in the shift of both difference curves of Fig. 1 toward longer wavelengths. Moreover, the effect of such product interference would have been especially severe in the case of a visual pi_gment absorbing in the blue as does the green rod pigment. The attempt to carry out a partial bleaching analysis in the presence of hydroxylamine revealed that
Fig. 1. The presence of the green rod pigment as revealed by partial bleaching analysis. Filled circles and solid line represent the optical density change due to exposure of a higitonin extract of bullfrog out& se_gments to 600-nm lieht for 17 hr 35 mm. Filled sauares and dashed line reoresent the optical density change due to subsequent expbsure of the same sample to 470-nm light for 2 hr. Positive values represent optical density decreases; negative values optical density increases.
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Fig. 2. Destruction of the green rod pigment by hydroxylamine. Curve 1 represents the optical density change due to addition of @l ml of 0.2 M neutralized hydroxylamine to 1,Oml of a digitonin extract of bullfrog retinas. Curve 2 represents the optical density change due to exposure of the sample to 6%nm light for I80 min. Curve 3 rep resents the optical density chang.e due to additional exposure of the sample to 60&m hght for 60 min. Curve 4 represents the optical density change due to additional exposure of the sample to white light for 20 min. Curve 5 represents the total optical density change due to all light exposures. The data shown represent the average of five different experiments with aliquots of the same extract. All readings have been corrected for dilution due to the addition of hydroxylamine by multiplying by 1.1. Positive values represent optical density decreases; negative values optical density increases.
the green rod pigment of the bullfrog is sensitive to that reagent. Indeed, addition of 0.1 ml of 0.7 ,~1neutralized hydroxylamine to I.Oml of visual pigment destroyed ah of the green rod pigment in less than two hours. This was not unexpected since the green rod pigment of Ram femporarin (Reuter, 1966) and of Rma cancricuru (DartnaIl, 1967) were found to be destroyed by exposure to hydroxylam~e. The hydroxylamine sensitivity of the green rod pigment of the bullfrog is shown by the experiment illustrated in Fig. 2. Curve 1 represents the optical density change in a digitonin extract of bullfrog retinas following the addition of hydroxylamine. It is obvious that the reagent has destroyed a substance which absorbs maximally near 435 nm. Since the hydroxylamine, by combining with retinal, minimizes product interference, and since rhodopsin contamination should not be a factor in this procedure, the peak of this curve is a much better reflection of the true /.,,, of the green rod pigment than is the peak of the curve (A) shown in Fig. 1. The 435nm value obtained here is in agreement with the j,,,,, of the green rod pigment of Ram cmcricortl which was obtained by Dartnall (1967) using the same method. Curve 1 of Fig. 2 also shows that destruction of the green rod pigment with hydroxyiamine results in the formation of an oxime with maximum absorbance near 365 nm. This indicates that the green rod pigment of the bullfrog is based on retinal. Treatment of triton X-100 extracts (IO/;;in 0.1 M phosphate buffer, pH 7.4) with hydroxylamine yielded results similar to those described just above. Following the destruction of the green rod pigment with hydroxylamine, samples were partially bleached
to determine the pigment composition of the remaining photosensitive material. AS illustrated by curves 2, 3. 4 and 5 in Fig. 2. all difference curves obtained in the analysis. regardless of the wav-elength of the bleaching light. exhibited visual pigment maxima near 504nm and product maxima near 37Onm. Thus. it is reasonable to conclude that the extract of bullfrog retinas used in the experiment of Fig. 1 contained only rhodopsin (VPSO4,) in addition to the green rod pi_ment. No traces of porphyropsin (VP524?) were observed. Indeed, several different extracts were prepared at various times during the year (11 April, 17 May and 3 October) and analysis always revealed the presence only of rhodopsin and the green rod pigment. No porphyropsin was ever detected. Analysis. by exposure to h~droxyiami~e followed by partial bleaching. of a sample prepared from a group of retinas from which more than 95”; of the visual pigment was extracted revealed that the green rod pigment is present in the bullfrog retina in an amount (in terms of optical density at maximum) equal to 8.7”” that of rhodopsin. The extract which yielded the data of Fig. 2 actually contained green rod pigment with an optical density equal to about 11”; that of rhodopsin in the same extract. That value. however. is not an accurate representation of the relative percentage of green rod pigment in the bullfrog retina. This is so since no attempt was made
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Fig. 3. Comparison of the hydroxylamine destruction spectrum for the bullfrog’s green rod pigment with the nomogram. Filled circles and dashed line are the normalized data of curv-e 1 in Fig 3 and represent the hydroxylamine destruction spectrum obtained in the present study with digitonin extracts of whole bullfrog retinas. Open squares represent the data obtained here with a digitonin extract of bullfrog outer segments. Open triangles represent Dartnail’s (1967) hydroxylamine destruction spectrum for the green rod pi_ment of Rnnn cmerirorn. The thin solid line is the nomogram curve for a retinal based visual pigment absorbing maximally at 435 nm.
Research Note with that preparation to extract virtually all the visual pigment from the tissue. Exhaustive extraction of pig ment is necessary because it appears that the green rod pigent is preferentially extracted over rhodopsin. Thus, it was found in this study, as it was by Dartnall (1967) with Rana cancriuorn, that earlier extractions tended to contain higher percentages of green rod pigment than did later extractions. For example, with one digitonin preparation of whole retinas the first three extractions, which were pooled, provided a sample which contained (in terms of optical density at rn~~urn) 15-Z’/, green rod pigment. In contrast, the fourth, fifth and sixth extractions yielded amounts of 69. 4% and 5.8% respectively. In Fig. 3 the difference spectrum obtained by reaction of the bullfrog’s green rod pigment with hydroxylamine (filled circles) is compared with the spectrum obtained in the same manner by Dartnall (1967) for Runa cancriooru (open triangles) and with the Dartnall (1953) nomogram (solid thin line) for a retinal based visual pigment absorbing maximally at 435 nm. Although the two experimental curves agree we11with each other at shorter wavelengths, they are both depressed with respect to the shorter wavelength portion of the nomogram, presumably as a result of the product interference due to absorbance by the oxime. However, whereas the Rnna ccmcricorn curve is in excellent agreement with the VP435, nomogram at longer wavelengths, the bullfrog curve is significantly elevated. The reason for this deviation from the nomogram is not yet clear. However, it is quite possible that the samples contained another substance, in addition to the green rod pigment. which absorbs at longer wavelengths and which is also sensitive to hydroxyl~ine. Further study is necessary to identify the hypothetical substance but it is interesting to speculate that the bullfrog extracts contained a small amount of accessory cone pigment. The presumptive cone pigments of the chicken (Wald, 1958) and the pigeon (Bridges, 1962) are much more sensitive to hydroxylamine than are typical rhodopsins and it is suspected that this may be a characteristic of cone pigments in general. The bullfrog retina is known to contain accessory cones (Steinberg, 1973) and it is likely that their pigment absorbs maximally near 5OOmn, as is the case with Rann pipiens (Liebman and Entine, 1968). Such a visual pigment would be indistin~ishable from rhodopsin by partial bleaching analysis. Since the green rod pigment is known to have a greater regenerative capability than rhodopsin in the intact retina (Donner and Reuter, 1962; Reuter, 1966; Goldstein and Wolf, 1973) it was of interest to determine whether or not this is also the case in digitonin extracts. Accordingly, samples of bullfrog visual pigment extracts were completely bleached by exposure to white room light for 30 min at ZO’C, after which they were placed in the s~trophotometer at 20°C and their regeneration monitored. In Fig. 4 curve IA represents the optical density change which resulted from the initial exposure of the sample to white light. The difference spectrum that describes the optical density decrease is one typical of visual pigments with a maximum loss at about 507 nm. In contrast, curve 2B represents the material that regenerated after 10 min in the dark at 25°C. It is apparent that the maxi-
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Fig. 4. The regeneration of bullfrog visual pigments at 20°C. Curve 1A represents the optical density change due to initial exposure of a digitonin extract of whole retinas to white light for 30 min. Curve 2B represents the optical density change of the sample upon sitting in the dark for 10 min. Curve 3B represents the optical density change after 45 min in the dark. Curve 48 represents the optical density change after 80 min in the dark. Curve SB represents the optical density change due to a final 10 min bleach with white light. The data represent the average of four experiments. All measurements were made at 20°C. Positive values represent optical density decreases: negative values optical density increases.
mum increase in optical density is not near 557nm, but rather is in the vi&nip of 47Onm. Even if one assumes that a considerable amount of the regenerated material is isorh~ops~ &,,,487 nm) one cannot account for the position of the i,,,=,of the regenerated material without concludin! that regeneration of the green rod pigment is affectmg the curve to a degree far in excess of that expected solely on the basis of its relative concentration. Even after periods of 45 min (curve 3B) and 80 min (curve 4B) in the dark the regeneration curves do not resemble the initial bleaching curve (curve lA). The regeneration curves are greatly elevated in the shorter wavelength region, an elevation most likely due to regeneration of the green rod pigment. However, in contrast to the curve obtained after 10 min regeneration (curve 23) the maxima of curve 3B and curve 4B are no longer at 470 MI but are shifted to about 495 nm. Such a shift , m krnaxwould be expected if regeneration of the green rod pi_Fent had already neared completion but regeneration of rhodopsin, the overwhelmingly major component, were still occurring. That all of the material regenerated was photosensitive is shown by curve 5B which represents the absorbance change due to a final I5 min exposure to white room light following regeneration. Significantly, curve 53 is virtually the mirror image of curve 4B, the Iatter representing the material regenerated after 80 min in the dark. All of this leads to the conclusion that the green rod pigment regenerates in digitonin solutions at a greater rate than does rhodopsin. This is consistent with the results of the studies performed with intact retinas and, moreover, indicates that the greater regenerative capability of the green rod pigment is dependent, at
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1
one must conclude that the regenerability of rhodopsin and of the green rod pigment are equally aRiited by decreased temperature. Decreasing the temperature did indeed greatly reduce the rate of regeneration since it required 93 mm in the dark at S’C to produce the same increase in optical density (O.OSO.026 optical density units) at 470nm that required only 10 min at 1O.C. Ir is concluded from this work that the retina of the bullfrog does indeed contain a blue absorbing visual pigment and that the pigment is similar in its characteristics to the green rod pi_ements found in other species of frogs. Thus, the green rod pi_gment of the buttfrog has a i,,, of 435 i: 2 nm and is based on retinal. It is destroyed by hydroxylamine in a concentration which ordinarily does not affect frog rhodopsin. It is preferentially extracted over rhodopsin by digitonin. And. tinally. it regenerates in digitonin solution at a faster rate than does frog rhodopsin. rlc.~trotvietftrernrrli-The author wishes to thank Mr. John Krriuiand and .Ifr, Jack S. Gildar for their valuable technical assistance. REFERENCES
Fig. 5. The regeneration of bullfrog visual pigments at 5’C. Filled circles and dashed line represent the optical density change in a bleached digitonin extract of bullfrog retinas after sitting in the dark at 20°C for 10 min. The data are actuallv those of curve 2B in Fig. ;I followinz normalization at the maximum. .Actual optical density increase is 0.025 at the peak. Open circles represent the normalized optical density change in a bleached aliquot of the same sample after 93 min in the dark at 5’C. Actual optical density increase was 0.026 at the peak. Positive values represent optical density decreases: negative values optical density increases.
feast to a large degree, upon the structure of the visual pigment molecule rather than upon the morphology of the green rods. On the chance that the respective regeneration rates of rhodopsin and the green rod pi_ment might be differentially sensitive to temperature, severa regeneration experiments were carried out at 5’C rather than at 20°C. In these experiments the sample was exposed to white room light for 29 min at WC and for a fmal minute at 5°C. The sample was then returned to the sp~rophotometer cell compa~ment, maintained at YC, and its regeneration monitored. The results of a typical experiment, using an aliquot of the same sample used in the experiment of Fig. 4, are illustrated in Fig. 5, The filled squares represent the normalized change in optical density of the sample following 93 min in the dark at SC. The i,,, of the regenerated material is in the region of 470 nm and, therefore, similar to the i.,,, of the material regenerated after 10 min at 20°C (curve 1B, Fig. 4). In fact, the tilled circles and dashed line in Fig. 5 represent the data of curve ZB in Fig. 4 following normal~ation. Clearly. therefore, there is no significant difference in the two sets of normalized data and
Bridges C. D. B. (1962) Visual pigments of the pigeon (Coi~rnthrrlkitr). I ision Res. 2. 1%L37. Crescitelli F. (1972) The visual cells and visual p&xnts of the kertebrate eye. In Handbook of Se’nsor~ Ph~siu/ogy. Vol. VII. Pt. I. Phoruchtinristry of‘I,‘isiou (Edited by Dartnail H. J. A.). Springer. New York. DartnaIl H. J. A. (1952) Visual pigment 467. a photosensirite pigment present in tenth retinae. f. Pirysiol.. Land. 116. X7-259. Dartnall H. J. .A. (1953) The interpretation of spectral sensitit it)- curbes. Br. mcrl. Bu//. 9. 21--N,. Dxtnall H. J. A. (lY57) T/n, Ciwtrl Pi~gmtit~r.~ Wile!. New York. Darmall H. J. A. (1967) The visual pi_ment of the green rods. I.&ion Rzs. 7. t-i6. Denton E. J. and Wyllie J. H. (19.55)Study of the photosensitive pigments in the pink and green rods of the frog. J. Physiok, Land. 127, 51-89. . Donner K. 0. and Reutsr T. (1962) The spectral sensitivity and photopigment of the green rods in the frog’s retina. lision Rrs. 2. 357-372. Goldstein E. B. and Wolf B. M. (1973) Regeneration of the green-rod pigment in the isolated frog retina. p&ion Rrs. 13. 527-534. Liebman P. .A. and Entine G. (1968) Visual pigments of frog and tadpole (R~NI pipiens). l4sion Rrs. .8; 761-775. Rsuter T. (1966) The svnthesis of nhotoscnsitive uiarnents in rods of the frog retina. t&f& Rrs. 6. 15-38.’ Reuter T. E., White R. H. and Wafd G. (1971) Rhodopsin and porphyropsin fields in the adult bullfrog retina. J. gen. Phxsiol. 58, 351-371. Sillman A. J. (1974) Rapid dark adaptation in the frog cone. iision Res. 11, 1021-1027. Sillman A. J.. Owen CV. G. and Fernandn H. R. (1972) The eeneration of the late receptor potential: an eucitation-inhibition ohenomenon. !&ion Res. 12. 1519-1531. Steinberg R. H. (1973) Scanning electron microscopy of the bullfrog’s retina and pigment epithelium. Z. Zel[$xsch. 113. A5l-463. EVaId G. (1953) Retinal chemistry and the physiology- of Phys. Lab..
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London. pp. 7-61.
OF THE BULLFROG
J. SILLsUN’
.&SOLD
Department of Biology. University of Pittsburgh, Pittsburgh. PA 15260, U.S.A. lRrceicen 9 July 1975)
In a study of rapid dark adaptation Sillman (1974) reported that the scotopic spectral sensitivity curve derived from the photoreceptors of the isolated. perfused bullfrog retina was much broader than that which should have been expected if only rhodopsin were involved. It was suggested that the elevation at the longer wavelengths mi_$tt be due, on the one hand, to cones which are acttve under scotopic conditions and, on the other hand, to the possible presence of porphyropsin (VP524,) a visual pinent which absorbs at longer wavelengths than bullfrog rhodopsin (VP504i) and which is sometimes present in the retina of the bullfrog (Reuter, White and Wald, 1971: Sillman, Owen and Femandez, 1972). It was also suggested that the elevation at the shorter wavelengths might be due, at least in part, to the presence of the green rod pigment which has been observed in the retinas of several other ranids and which absorbs maximally in the region of 435 nm (Denton and Wyllie, 1955: Dartnall, 1957, 1967; Donner and Reuter, 1962: Reuter. 1966: Liebman and Entine. 1968. See Crescitelli, 1972, for a review.). Since green rods have been described in the bullfrog retina (Steinberg, 1973), it certainly is a logical assumption that this retina contains the blue absorbing pigment typical of those photoreceptors. Surprisingly. however. there is no report in the literature of the presence or absence of such a visual pigment in the bullfrog retina, even though the bullfrog is a very common experimental animal in studies concerned with the visual system. The present work, therefore, was carried out to determine whether or not a blue absorbing photopigment does indeed exist in the retina of the bullfrog and, if so. what the properties are. Accordingly, an extract (2”; digitonin in 0.1 M phosphate buffer, pH 7.4) of bullfrog (Rana cnresbeiana) outer segments was subjected to a simple, two-step, partial bleaching analysis using the method of Dartnall (1952). Thus, the sample was exposed first to 600 nm light for 17 hr 35 min. a treatment which is expected to bleach nearly all visual pigment absorbing maximally at 500 nm or higher but which should leave untouched any visual pi_ment absorbing maximally in the blue region of the spectrum. After the lengthy exposure to 600nm light the sample was then exposed for 2 hr to light of 470nm, a wavelength which is expected to bleach any visual pigment absorbing in the blue. The results, displayed as difference spectra, are illustrated in Fig. 1. The filled circles (B) represent the change resulting from the exposure to 600nm light. ’ Present address: Dept. of Animal Physiology. University of California, Davis, CA 95616, U.S.A. 421
It is apparent that this red light bleached out pigment absorbing maximally near 5OOnm. In contrast, the subsequent exposure of the sample to 470nm light resulted in a decrease in optical density (A) which was maximal not in the region of 5OOnm, but close to 45Onm. From this it may be concluded that the extract contained a pigment absorbing in the blue region of the spectrum which is probably the visual pigment contained within the green rods. However, for two reasons the filled squares of Fig. 1 cannot accurately reflect the true spectral absorbance curve of the bullfrog’s green rod pigment. First, it is not unlikely that some small amount of rhodopsin remained in the sample even after the lengthy exposure to 600 nm light. This residual rhodopsin (VP504i) would be destroyed together with the green rod pigment upon exposure of the sample to 470nm light and, therefore, would tend to shift the resulting difference curve toward longer wavelengths. Second, and much more important, since the analysis was performed in the absence of hydroxylamine, product interference undoubtedly resulted in the shift of both difference curves of Fig. 1 toward longer wavelengths. Moreover, the effect of such product interference would have been especially severe in the case of a visual pi_gment absorbing in the blue as does the green rod pigment. The attempt to carry out a partial bleaching analysis in the presence of hydroxylamine revealed that
Fig. 1. The presence of the green rod pigment as revealed by partial bleaching analysis. Filled circles and solid line represent the optical density change due to exposure of a higitonin extract of bullfrog out& se_gments to 600-nm lieht for 17 hr 35 mm. Filled sauares and dashed line reoresent the optical density change due to subsequent expbsure of the same sample to 470-nm light for 2 hr. Positive values represent optical density decreases; negative values optical density increases.
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Fig. 2. Destruction of the green rod pigment by hydroxylamine. Curve 1 represents the optical density change due to addition of @l ml of 0.2 M neutralized hydroxylamine to 1,Oml of a digitonin extract of bullfrog retinas. Curve 2 represents the optical density change due to exposure of the sample to 6%nm light for I80 min. Curve 3 rep resents the optical density chang.e due to additional exposure of the sample to 60&m hght for 60 min. Curve 4 represents the optical density change due to additional exposure of the sample to white light for 20 min. Curve 5 represents the total optical density change due to all light exposures. The data shown represent the average of five different experiments with aliquots of the same extract. All readings have been corrected for dilution due to the addition of hydroxylamine by multiplying by 1.1. Positive values represent optical density decreases; negative values optical density increases.
the green rod pigment of the bullfrog is sensitive to that reagent. Indeed, addition of 0.1 ml of 0.7 ,~1neutralized hydroxylamine to I.Oml of visual pigment destroyed ah of the green rod pigment in less than two hours. This was not unexpected since the green rod pigment of Ram femporarin (Reuter, 1966) and of Rma cancricuru (DartnaIl, 1967) were found to be destroyed by exposure to hydroxylam~e. The hydroxylamine sensitivity of the green rod pigment of the bullfrog is shown by the experiment illustrated in Fig. 2. Curve 1 represents the optical density change in a digitonin extract of bullfrog retinas following the addition of hydroxylamine. It is obvious that the reagent has destroyed a substance which absorbs maximally near 435 nm. Since the hydroxylamine, by combining with retinal, minimizes product interference, and since rhodopsin contamination should not be a factor in this procedure, the peak of this curve is a much better reflection of the true /.,,, of the green rod pigment than is the peak of the curve (A) shown in Fig. 1. The 435nm value obtained here is in agreement with the j,,,,, of the green rod pigment of Ram cmcricortl which was obtained by Dartnall (1967) using the same method. Curve 1 of Fig. 2 also shows that destruction of the green rod pigment with hydroxyiamine results in the formation of an oxime with maximum absorbance near 365 nm. This indicates that the green rod pigment of the bullfrog is based on retinal. Treatment of triton X-100 extracts (IO/;;in 0.1 M phosphate buffer, pH 7.4) with hydroxylamine yielded results similar to those described just above. Following the destruction of the green rod pigment with hydroxylamine, samples were partially bleached
to determine the pigment composition of the remaining photosensitive material. AS illustrated by curves 2, 3. 4 and 5 in Fig. 2. all difference curves obtained in the analysis. regardless of the wav-elength of the bleaching light. exhibited visual pigment maxima near 504nm and product maxima near 37Onm. Thus. it is reasonable to conclude that the extract of bullfrog retinas used in the experiment of Fig. 1 contained only rhodopsin (VPSO4,) in addition to the green rod pi_ment. No traces of porphyropsin (VP524?) were observed. Indeed, several different extracts were prepared at various times during the year (11 April, 17 May and 3 October) and analysis always revealed the presence only of rhodopsin and the green rod pigment. No porphyropsin was ever detected. Analysis. by exposure to h~droxyiami~e followed by partial bleaching. of a sample prepared from a group of retinas from which more than 95”; of the visual pigment was extracted revealed that the green rod pigment is present in the bullfrog retina in an amount (in terms of optical density at maximum) equal to 8.7”” that of rhodopsin. The extract which yielded the data of Fig. 2 actually contained green rod pigment with an optical density equal to about 11”; that of rhodopsin in the same extract. That value. however. is not an accurate representation of the relative percentage of green rod pigment in the bullfrog retina. This is so since no attempt was made
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Fig. 3. Comparison of the hydroxylamine destruction spectrum for the bullfrog’s green rod pigment with the nomogram. Filled circles and dashed line are the normalized data of curv-e 1 in Fig 3 and represent the hydroxylamine destruction spectrum obtained in the present study with digitonin extracts of whole bullfrog retinas. Open squares represent the data obtained here with a digitonin extract of bullfrog outer segments. Open triangles represent Dartnail’s (1967) hydroxylamine destruction spectrum for the green rod pi_ment of Rnnn cmerirorn. The thin solid line is the nomogram curve for a retinal based visual pigment absorbing maximally at 435 nm.
Research Note with that preparation to extract virtually all the visual pigment from the tissue. Exhaustive extraction of pig ment is necessary because it appears that the green rod pigent is preferentially extracted over rhodopsin. Thus, it was found in this study, as it was by Dartnall (1967) with Rana cancriuorn, that earlier extractions tended to contain higher percentages of green rod pigment than did later extractions. For example, with one digitonin preparation of whole retinas the first three extractions, which were pooled, provided a sample which contained (in terms of optical density at rn~~urn) 15-Z’/, green rod pigment. In contrast, the fourth, fifth and sixth extractions yielded amounts of 69. 4% and 5.8% respectively. In Fig. 3 the difference spectrum obtained by reaction of the bullfrog’s green rod pigment with hydroxylamine (filled circles) is compared with the spectrum obtained in the same manner by Dartnall (1967) for Runa cancriooru (open triangles) and with the Dartnall (1953) nomogram (solid thin line) for a retinal based visual pigment absorbing maximally at 435 nm. Although the two experimental curves agree we11with each other at shorter wavelengths, they are both depressed with respect to the shorter wavelength portion of the nomogram, presumably as a result of the product interference due to absorbance by the oxime. However, whereas the Rnna ccmcricorn curve is in excellent agreement with the VP435, nomogram at longer wavelengths, the bullfrog curve is significantly elevated. The reason for this deviation from the nomogram is not yet clear. However, it is quite possible that the samples contained another substance, in addition to the green rod pigment. which absorbs at longer wavelengths and which is also sensitive to hydroxyl~ine. Further study is necessary to identify the hypothetical substance but it is interesting to speculate that the bullfrog extracts contained a small amount of accessory cone pigment. The presumptive cone pigments of the chicken (Wald, 1958) and the pigeon (Bridges, 1962) are much more sensitive to hydroxylamine than are typical rhodopsins and it is suspected that this may be a characteristic of cone pigments in general. The bullfrog retina is known to contain accessory cones (Steinberg, 1973) and it is likely that their pigment absorbs maximally near 5OOmn, as is the case with Rann pipiens (Liebman and Entine, 1968). Such a visual pigment would be indistin~ishable from rhodopsin by partial bleaching analysis. Since the green rod pigment is known to have a greater regenerative capability than rhodopsin in the intact retina (Donner and Reuter, 1962; Reuter, 1966; Goldstein and Wolf, 1973) it was of interest to determine whether or not this is also the case in digitonin extracts. Accordingly, samples of bullfrog visual pigment extracts were completely bleached by exposure to white room light for 30 min at ZO’C, after which they were placed in the s~trophotometer at 20°C and their regeneration monitored. In Fig. 4 curve IA represents the optical density change which resulted from the initial exposure of the sample to white light. The difference spectrum that describes the optical density decrease is one typical of visual pigments with a maximum loss at about 507 nm. In contrast, curve 2B represents the material that regenerated after 10 min in the dark at 25°C. It is apparent that the maxi-
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Fig. 4. The regeneration of bullfrog visual pigments at 20°C. Curve 1A represents the optical density change due to initial exposure of a digitonin extract of whole retinas to white light for 30 min. Curve 2B represents the optical density change of the sample upon sitting in the dark for 10 min. Curve 3B represents the optical density change after 45 min in the dark. Curve 48 represents the optical density change after 80 min in the dark. Curve SB represents the optical density change due to a final 10 min bleach with white light. The data represent the average of four experiments. All measurements were made at 20°C. Positive values represent optical density decreases: negative values optical density increases.
mum increase in optical density is not near 557nm, but rather is in the vi&nip of 47Onm. Even if one assumes that a considerable amount of the regenerated material is isorh~ops~ &,,,487 nm) one cannot account for the position of the i,,,=,of the regenerated material without concludin! that regeneration of the green rod pigment is affectmg the curve to a degree far in excess of that expected solely on the basis of its relative concentration. Even after periods of 45 min (curve 3B) and 80 min (curve 4B) in the dark the regeneration curves do not resemble the initial bleaching curve (curve lA). The regeneration curves are greatly elevated in the shorter wavelength region, an elevation most likely due to regeneration of the green rod pigment. However, in contrast to the curve obtained after 10 min regeneration (curve 23) the maxima of curve 3B and curve 4B are no longer at 470 MI but are shifted to about 495 nm. Such a shift , m krnaxwould be expected if regeneration of the green rod pi_Fent had already neared completion but regeneration of rhodopsin, the overwhelmingly major component, were still occurring. That all of the material regenerated was photosensitive is shown by curve 5B which represents the absorbance change due to a final I5 min exposure to white room light following regeneration. Significantly, curve 53 is virtually the mirror image of curve 4B, the Iatter representing the material regenerated after 80 min in the dark. All of this leads to the conclusion that the green rod pigment regenerates in digitonin solutions at a greater rate than does rhodopsin. This is consistent with the results of the studies performed with intact retinas and, moreover, indicates that the greater regenerative capability of the green rod pigment is dependent, at
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one must conclude that the regenerability of rhodopsin and of the green rod pigment are equally aRiited by decreased temperature. Decreasing the temperature did indeed greatly reduce the rate of regeneration since it required 93 mm in the dark at S’C to produce the same increase in optical density (O.OSO.026 optical density units) at 470nm that required only 10 min at 1O.C. Ir is concluded from this work that the retina of the bullfrog does indeed contain a blue absorbing visual pigment and that the pigment is similar in its characteristics to the green rod pi_ements found in other species of frogs. Thus, the green rod pi_gment of the buttfrog has a i,,, of 435 i: 2 nm and is based on retinal. It is destroyed by hydroxylamine in a concentration which ordinarily does not affect frog rhodopsin. It is preferentially extracted over rhodopsin by digitonin. And. tinally. it regenerates in digitonin solution at a faster rate than does frog rhodopsin. rlc.~trotvietftrernrrli-The author wishes to thank Mr. John Krriuiand and .Ifr, Jack S. Gildar for their valuable technical assistance. REFERENCES
Fig. 5. The regeneration of bullfrog visual pigments at 5’C. Filled circles and dashed line represent the optical density change in a bleached digitonin extract of bullfrog retinas after sitting in the dark at 20°C for 10 min. The data are actuallv those of curve 2B in Fig. ;I followinz normalization at the maximum. .Actual optical density increase is 0.025 at the peak. Open circles represent the normalized optical density change in a bleached aliquot of the same sample after 93 min in the dark at 5’C. Actual optical density increase was 0.026 at the peak. Positive values represent optical density decreases: negative values optical density increases.
feast to a large degree, upon the structure of the visual pigment molecule rather than upon the morphology of the green rods. On the chance that the respective regeneration rates of rhodopsin and the green rod pi_ment might be differentially sensitive to temperature, severa regeneration experiments were carried out at 5’C rather than at 20°C. In these experiments the sample was exposed to white room light for 29 min at WC and for a fmal minute at 5°C. The sample was then returned to the sp~rophotometer cell compa~ment, maintained at YC, and its regeneration monitored. The results of a typical experiment, using an aliquot of the same sample used in the experiment of Fig. 4, are illustrated in Fig. 5, The filled squares represent the normalized change in optical density of the sample following 93 min in the dark at SC. The i,,, of the regenerated material is in the region of 470 nm and, therefore, similar to the i.,,, of the material regenerated after 10 min at 20°C (curve 1B, Fig. 4). In fact, the tilled circles and dashed line in Fig. 5 represent the data of curve ZB in Fig. 4 following normal~ation. Clearly. therefore, there is no significant difference in the two sets of normalized data and
Bridges C. D. B. (1962) Visual pigments of the pigeon (Coi~rnthrrlkitr). I ision Res. 2. 1%L37. Crescitelli F. (1972) The visual cells and visual p&xnts of the kertebrate eye. In Handbook of Se’nsor~ Ph~siu/ogy. Vol. VII. Pt. I. Phoruchtinristry of‘I,‘isiou (Edited by Dartnail H. J. A.). Springer. New York. DartnaIl H. J. A. (1952) Visual pigment 467. a photosensirite pigment present in tenth retinae. f. Pirysiol.. Land. 116. X7-259. Dartnall H. J. .A. (1953) The interpretation of spectral sensitit it)- curbes. Br. mcrl. Bu//. 9. 21--N,. Dxtnall H. J. A. (lY57) T/n, Ciwtrl Pi~gmtit~r.~ Wile!. New York. Darmall H. J. A. (1967) The visual pi_ment of the green rods. I.&ion Rzs. 7. t-i6. Denton E. J. and Wyllie J. H. (19.55)Study of the photosensitive pigments in the pink and green rods of the frog. J. Physiok, Land. 127, 51-89. . Donner K. 0. and Reutsr T. (1962) The spectral sensitivity and photopigment of the green rods in the frog’s retina. lision Rrs. 2. 357-372. Goldstein E. B. and Wolf B. M. (1973) Regeneration of the green-rod pigment in the isolated frog retina. p&ion Rrs. 13. 527-534. Liebman P. .A. and Entine G. (1968) Visual pigments of frog and tadpole (R~NI pipiens). l4sion Rrs. .8; 761-775. Rsuter T. (1966) The svnthesis of nhotoscnsitive uiarnents in rods of the frog retina. t&f& Rrs. 6. 15-38.’ Reuter T. E., White R. H. and Wafd G. (1971) Rhodopsin and porphyropsin fields in the adult bullfrog retina. J. gen. Phxsiol. 58, 351-371. Sillman A. J. (1974) Rapid dark adaptation in the frog cone. iision Res. 11, 1021-1027. Sillman A. J.. Owen CV. G. and Fernandn H. R. (1972) The eeneration of the late receptor potential: an eucitation-inhibition ohenomenon. !&ion Res. 12. 1519-1531. Steinberg R. H. (1973) Scanning electron microscopy of the bullfrog’s retina and pigment epithelium. Z. Zel[$xsch. 113. A5l-463. EVaId G. (1953) Retinal chemistry and the physiology- of Phys. Lab..
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London. pp. 7-61.