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Modified rhodopsin in the pigment epithelium?
Vision Rcs. Vol. 13, pp. 477480. Perpmxm PICSS1973. Printed in GMt
Britain.
RESEARCH .NOTE MODIFIED
RHODOPSIN
IN THE PIGMENT
EPITHELIUM?
Laboratoryof VisionResearch,NationalEye Institute,NationalInstituteof Health,Bethesda, Maryland20014,U.S.A. (Received 1 May 1972; in revisedform 6 Jury 1972)
RECENTautoradiographic studies have demonstrated that proteins of the receptor outer segment are continuously renewed in the inner segment and removed at the apical region of the outer segment by the action of “phagosomes”, presumably a type of lysosomes present in the pigment epithelium (DROZ, 1963 ; YOUNGand DROZ, 1968 ; YOUNGand BOK, 1969). During this renewal process, rhodopsin is also synthesized and degraded (HALL, BOK and BACHARACH,1969). If the phagocytic digestion of disc membranes is responsible for the decomposition of rhodopsin, it is expected that breakdown products of rhodopsin may be found in the pigment epithelial cells. I report in this paper that photosensitive pigments which are not rhodopsin can be spectrally detected in the pigment epithelium of the bovine eye. Fresh bovine eyes were purchased from a local slaughterhouse. The eyes (about 50) were opened in dim red light and the pigment epithelium layer was carefully separated from the retina, washed twice with M/15 potassium phosphate buffer, pH 6.5 and ground gently by hand in 40% sucrose-buffer (pH 6.5). The tissue homogenate was filtered through a layer of cheesecloth, diluted with buffer to approximately 8.6 per cent (O-25 M) sucrose and centrifuged at 12,000 g for 15 min. In some experiments, the solution was diluted to 4 % sucrose and centrifuged at 25,OOOg to increase the yield of sediment. The sediment was washed three times with buffer and water to remove hemoglobin. In later experiments, the crude filtrate in 40 % sucrose was spun at 300 g for 5 min to remove dark pigment granules before the solution was diluted and centrifuged. The sediment showed a distinct acid phosphatase activity, a marker enzyme of lysosomes, when assayed with ~-glycerophosphate as substrate (HIRSCHHORN, HIRSCHHORN and W~SSMA~, 1971). Using hi&chemical techniques, MARSKUL (1970) has shown that a positive acid phosphatase reaction is obtained from the phagocytic inclusion bodies of rabbit pigment epithelium. The sediment thus prepared was then extracted with 5 ml of 1% Emulphogene BC 720 (GAF Corp., New York) buffered at pH 6.5 and centrifuged at 100,000 g for 60 min, and the supernatant was spectrally examined with a Gary 14 recording spectrometer. The crude lysosomal fraction could be further purified by the method of B-N, BACHand DZODZOE(1971). However, extensive purification of phagosomes was not usually attempted in the present investigation since the yield of purified phagosomes was low. As shown in Fig. l-1, the spectrum of the extract suggested the presence of two components; one with an absorption maximum at 498 nm (rhodopsin) and the other with a maximum between 440 and 450 nm. By subtracting the spectrum of rhodopsin from the complex spectrum, the spectrum of the second component was isolated (see inset). Based 477
478
RESEARCH NOTE
0.7 l-
0
I
400
I 450
I
1
I
500
550
600
Wavelength, @4r
0
I
650
m-n
-f-- 0
! 400
1
450
I
500
Wavelength,
1
1
I
550
603
650
nm
FIG. 1. Spectral identification of PhdOin the pigment epithelium extract. A mixture of 3-Oml extract and O-2ml of 05 M hydroxylamine (pH 6.5) was poured into two cuvettes. One of the cuvettes was exposed to room light for 10 min and the difference (unbleached vs. bleached) spectrum was recorded (1-I). The unbleached sample was then exposed to light and a base line (bleached vs. bleached) was recorded. Assuming that the base lime should have no light absorbance over a wavelength range from 400 to 650 nm, a corrected spectrum was prepared. A spectrum similarly prepared with purified rhodopsin (SCHW, LEWIS,IRREVERRE and STONE, 1969) was superimposed on the spectrum. From these two spectra, the spectrum of Pa0 was obtained (see inset). For comparison, the difference (unbleached vs. bleached) spectrum of retinal extracts recorded in a similar manner is also shown (I-II).
on the absorption maximum at 440 nm, the component was designated P,,,. The pigment appeared to be stable in a pH range from 6 to 7.5 and it is unlikely that Pa4e is an artifact possibly caused by a slight change in the pH. Since no absorption band was found at 440 nm after the extract was bleached by light, P,,, was considered to be a photosensitive pigment. The absorption spectrum of P,,, was not modified immediately after the addition of NH*OH; on standing in NHzOH at 2°C for a few days it was gradually decomposed. Assuming that the molar extinction coefficients of rhodopsin and Pd4,, at the absorption maxima are the same (eM = 40,000), concentrations of rhodopsin and P,,, were estimated to be 8.7 run and 0.6 nm per 50 bovine eyes, respectively. The sum of these pigments in the extract accounted for approximately 20 per cent of the total retinal concentration of the pigment epithelium (approx. 50 nm per 50 eyes). It is not clear whether rhodopsin is actually present in the pigment epithelium or it is derived from contaminating outer segments. However, the fact that P,,, was found only in the extract of the pigment epithelium, while extracts similarly prepared from retinas did not contain the pigment (see Fig. l-11) strongly
479
RESEARCHNOTE
Wavelength,
nm
Fro. 2. Spectral separation of a 415-nm absorbing component. Absorption spectra were recorded on unbleached extracts against 1% Emuiphogene in MflS potassium phosphate
bufkr, pH 6.5. The spectrum 0- - - 0 was recorded after the addition of O-1ml of buffer to the pig&& epitbeli&n extract (1.4 ml), and the spectrum e -0 wastakenaftero*1ml of 03 M hydroxylamine (pH 6.5) was added to 1.4 ml of the pigment epithelium extract. The 415nm absorption band was separated from these spectra (see inset).
suggests the unique distribution of P440 in the pigment epithelium. Due to low concentrations of PbhOand because of interference by rhodopsin, it is not possible to obtain quantitative data on the partial bleaching of PbbO.Both rhodopsin and Ph4* appeared to be associated with a relatively labile structure since tissue homogenization by blending markedly reduced the yield of these pigments (l-3 nm of rhodopsin and 0.3 nm of PbbOper 50 eyes). Berman and Bach (personal communication) have found that the phagosomes of bovine pigment epithelium are readily ruptured by mechanical agitation. Although the concentration of Pd4,, varied depending on the method of preparation, the component was detected in all samples analyzed. Pd4,, is somewhat similar to an acid-denatured derivative of rhodopsin which has an absorption maximum at 440 nm (IWO, Suzuer, AZUMAand SEKOGUTI,1968). Therefore, it is interesting to see ifPhdO can be formed from membrane-bound rhodopsin by the action of phagosomal acid-hydrolases. As a preliminary experiment to test the possibility, the pigment epithelium was suspended in 0.2 M citric buffer, pH 4.8 and incubated for 4 hr at 35”C, and extracts from the incubated epithelium were compared with those from the control stored at 3°C at pH 4.8 or 6.5. The concentration of Pd4,, was not increased after incubation of the epithelial tissue at 35°C. Thus, it is yet to be determined whether or not PedO is indeed a degradation product of rhodopsin. In addition to PebO, another component having an absorption maximum at 415 nm
RESEARCH NOTE
480
was detected (Fig. 2). This component appeared to be photosensitive because the 415nm band vanished completely after bleaching. This pigment was rapidly decomposed by the addition of NHzOH or by prolonged storage at 3°C. Due to the lability of the component, the concentration of the component was not quantitatively determined. However, it was observed in all preparations examined. When spectra are recorded in the presence of NH,OH, this component is not detected. This is the reason why the component is absent in the spectrum of Fig. 1. The ,415-nm component can not be hemoglobin nor cytochrome P-450 (SHICHI, 1969) because its spectrum was not affected by carbon monoxide and the extract did not contain any appreciable amount of haematin as determined by preparing the pyridine haemochrome (SHICHI and HACKETT, 1962). Whether or not this component is related to rhodopsin
remains
to be investigated.
Acknowle&ernenrs--I thank Dr. E. R. BERMAN for sending me reprints and manuscripts concerning the preparation of pigment epithelial phagosomes and Miss J. DERRfor her technical assistance. REFERENCES BERMAN,E. R., BACH,G. and DZODZOE,Y. (1971). Distribution of hydrolytic enzymes in sub-cellular fractions of pigment epithelial cells. Israel J. Chem. 9, 11 BC. DROZ,B. (1963). Dynamic condition of proteins in the visual cells of rats and mice as shown by radioautography with labeled amino acids. Anat. Rec. 145, 157-167. HALL,M. 0.. EOK, D. and BACHARACH, A. D. E. (1969). Biosynthesis and assembly of the rod outer segment membrane system. Formation and fate of visual pigment in the frog retina. J. molec. Biof. 4!$387-406. G. (1967). Appearance of hydrolase rich granules in HXRSCHHORN, R., HIRXXHORN,K. and WEISSMANN, human lymphocytes induced by phytohemagglutinin and antigens. Blood 30,84-102. KITO, Y., SUSUKI,T., A~UMA,M. and SEKOOVTI, Y. (1968). Absorption spectrum of rhodopsin denatured with acid. Nature, Land. 218,955-956. MARQULL,J. (1970). Acid phosphatase activity in the retinal pigment epithelium. Vision Res. 10, 821-824. SHICHI,H. (1969). Microsomal electron transfer system of bovine retinal pigment epithelium. Exprl Eye Res. 8, 60-68. SHICHI,H. and HACKETT,D. P. (1962). Studies on the b-type cytochromes from mung bean seedlings. II. Some. properties of cytochromes b-555 and b-561. J. Biol. Chem. 237,2959-2964. E, F. and STONE,A. L. (1969). Biochemistry of visual pigments. I. PurificaSHICHI,H., LEWIS,M. S., IRREV~RR tion and properties of bovine rhodopsin. J. Biol. Chem. 244.529-536. YOUNG R. W. and BOK, D. (1969). Participation of the retinal pigment epithelium in the rod outer segment renewal process. J. Cell Biol. 42, 392-403. YOUNO, R. W. and DROZ, B. (1968). The renewal of protein in retinal rods and cones. J. Cell Biol. 39, 169-184.
Britain.
RESEARCH .NOTE MODIFIED
RHODOPSIN
IN THE PIGMENT
EPITHELIUM?
Laboratoryof VisionResearch,NationalEye Institute,NationalInstituteof Health,Bethesda, Maryland20014,U.S.A. (Received 1 May 1972; in revisedform 6 Jury 1972)
RECENTautoradiographic studies have demonstrated that proteins of the receptor outer segment are continuously renewed in the inner segment and removed at the apical region of the outer segment by the action of “phagosomes”, presumably a type of lysosomes present in the pigment epithelium (DROZ, 1963 ; YOUNGand DROZ, 1968 ; YOUNGand BOK, 1969). During this renewal process, rhodopsin is also synthesized and degraded (HALL, BOK and BACHARACH,1969). If the phagocytic digestion of disc membranes is responsible for the decomposition of rhodopsin, it is expected that breakdown products of rhodopsin may be found in the pigment epithelial cells. I report in this paper that photosensitive pigments which are not rhodopsin can be spectrally detected in the pigment epithelium of the bovine eye. Fresh bovine eyes were purchased from a local slaughterhouse. The eyes (about 50) were opened in dim red light and the pigment epithelium layer was carefully separated from the retina, washed twice with M/15 potassium phosphate buffer, pH 6.5 and ground gently by hand in 40% sucrose-buffer (pH 6.5). The tissue homogenate was filtered through a layer of cheesecloth, diluted with buffer to approximately 8.6 per cent (O-25 M) sucrose and centrifuged at 12,000 g for 15 min. In some experiments, the solution was diluted to 4 % sucrose and centrifuged at 25,OOOg to increase the yield of sediment. The sediment was washed three times with buffer and water to remove hemoglobin. In later experiments, the crude filtrate in 40 % sucrose was spun at 300 g for 5 min to remove dark pigment granules before the solution was diluted and centrifuged. The sediment showed a distinct acid phosphatase activity, a marker enzyme of lysosomes, when assayed with ~-glycerophosphate as substrate (HIRSCHHORN, HIRSCHHORN and W~SSMA~, 1971). Using hi&chemical techniques, MARSKUL (1970) has shown that a positive acid phosphatase reaction is obtained from the phagocytic inclusion bodies of rabbit pigment epithelium. The sediment thus prepared was then extracted with 5 ml of 1% Emulphogene BC 720 (GAF Corp., New York) buffered at pH 6.5 and centrifuged at 100,000 g for 60 min, and the supernatant was spectrally examined with a Gary 14 recording spectrometer. The crude lysosomal fraction could be further purified by the method of B-N, BACHand DZODZOE(1971). However, extensive purification of phagosomes was not usually attempted in the present investigation since the yield of purified phagosomes was low. As shown in Fig. l-1, the spectrum of the extract suggested the presence of two components; one with an absorption maximum at 498 nm (rhodopsin) and the other with a maximum between 440 and 450 nm. By subtracting the spectrum of rhodopsin from the complex spectrum, the spectrum of the second component was isolated (see inset). Based 477
478
RESEARCH NOTE
0.7 l-
0
I
400
I 450
I
1
I
500
550
600
Wavelength, @4r
0
I
650
m-n
-f-- 0
! 400
1
450
I
500
Wavelength,
1
1
I
550
603
650
nm
FIG. 1. Spectral identification of PhdOin the pigment epithelium extract. A mixture of 3-Oml extract and O-2ml of 05 M hydroxylamine (pH 6.5) was poured into two cuvettes. One of the cuvettes was exposed to room light for 10 min and the difference (unbleached vs. bleached) spectrum was recorded (1-I). The unbleached sample was then exposed to light and a base line (bleached vs. bleached) was recorded. Assuming that the base lime should have no light absorbance over a wavelength range from 400 to 650 nm, a corrected spectrum was prepared. A spectrum similarly prepared with purified rhodopsin (SCHW, LEWIS,IRREVERRE and STONE, 1969) was superimposed on the spectrum. From these two spectra, the spectrum of Pa0 was obtained (see inset). For comparison, the difference (unbleached vs. bleached) spectrum of retinal extracts recorded in a similar manner is also shown (I-II).
on the absorption maximum at 440 nm, the component was designated P,,,. The pigment appeared to be stable in a pH range from 6 to 7.5 and it is unlikely that Pa4e is an artifact possibly caused by a slight change in the pH. Since no absorption band was found at 440 nm after the extract was bleached by light, P,,, was considered to be a photosensitive pigment. The absorption spectrum of P,,, was not modified immediately after the addition of NH*OH; on standing in NHzOH at 2°C for a few days it was gradually decomposed. Assuming that the molar extinction coefficients of rhodopsin and Pd4,, at the absorption maxima are the same (eM = 40,000), concentrations of rhodopsin and P,,, were estimated to be 8.7 run and 0.6 nm per 50 bovine eyes, respectively. The sum of these pigments in the extract accounted for approximately 20 per cent of the total retinal concentration of the pigment epithelium (approx. 50 nm per 50 eyes). It is not clear whether rhodopsin is actually present in the pigment epithelium or it is derived from contaminating outer segments. However, the fact that P,,, was found only in the extract of the pigment epithelium, while extracts similarly prepared from retinas did not contain the pigment (see Fig. l-11) strongly
479
RESEARCHNOTE
Wavelength,
nm
Fro. 2. Spectral separation of a 415-nm absorbing component. Absorption spectra were recorded on unbleached extracts against 1% Emuiphogene in MflS potassium phosphate
bufkr, pH 6.5. The spectrum 0- - - 0 was recorded after the addition of O-1ml of buffer to the pig&& epitbeli&n extract (1.4 ml), and the spectrum e -0 wastakenaftero*1ml of 03 M hydroxylamine (pH 6.5) was added to 1.4 ml of the pigment epithelium extract. The 415nm absorption band was separated from these spectra (see inset).
suggests the unique distribution of P440 in the pigment epithelium. Due to low concentrations of PbhOand because of interference by rhodopsin, it is not possible to obtain quantitative data on the partial bleaching of PbbO.Both rhodopsin and Ph4* appeared to be associated with a relatively labile structure since tissue homogenization by blending markedly reduced the yield of these pigments (l-3 nm of rhodopsin and 0.3 nm of PbbOper 50 eyes). Berman and Bach (personal communication) have found that the phagosomes of bovine pigment epithelium are readily ruptured by mechanical agitation. Although the concentration of Pd4,, varied depending on the method of preparation, the component was detected in all samples analyzed. Pd4,, is somewhat similar to an acid-denatured derivative of rhodopsin which has an absorption maximum at 440 nm (IWO, Suzuer, AZUMAand SEKOGUTI,1968). Therefore, it is interesting to see ifPhdO can be formed from membrane-bound rhodopsin by the action of phagosomal acid-hydrolases. As a preliminary experiment to test the possibility, the pigment epithelium was suspended in 0.2 M citric buffer, pH 4.8 and incubated for 4 hr at 35”C, and extracts from the incubated epithelium were compared with those from the control stored at 3°C at pH 4.8 or 6.5. The concentration of Pd4,, was not increased after incubation of the epithelial tissue at 35°C. Thus, it is yet to be determined whether or not PedO is indeed a degradation product of rhodopsin. In addition to PebO, another component having an absorption maximum at 415 nm
RESEARCH NOTE
480
was detected (Fig. 2). This component appeared to be photosensitive because the 415nm band vanished completely after bleaching. This pigment was rapidly decomposed by the addition of NHzOH or by prolonged storage at 3°C. Due to the lability of the component, the concentration of the component was not quantitatively determined. However, it was observed in all preparations examined. When spectra are recorded in the presence of NH,OH, this component is not detected. This is the reason why the component is absent in the spectrum of Fig. 1. The ,415-nm component can not be hemoglobin nor cytochrome P-450 (SHICHI, 1969) because its spectrum was not affected by carbon monoxide and the extract did not contain any appreciable amount of haematin as determined by preparing the pyridine haemochrome (SHICHI and HACKETT, 1962). Whether or not this component is related to rhodopsin
remains
to be investigated.
Acknowle&ernenrs--I thank Dr. E. R. BERMAN for sending me reprints and manuscripts concerning the preparation of pigment epithelial phagosomes and Miss J. DERRfor her technical assistance. REFERENCES BERMAN,E. R., BACH,G. and DZODZOE,Y. (1971). Distribution of hydrolytic enzymes in sub-cellular fractions of pigment epithelial cells. Israel J. Chem. 9, 11 BC. DROZ,B. (1963). Dynamic condition of proteins in the visual cells of rats and mice as shown by radioautography with labeled amino acids. Anat. Rec. 145, 157-167. HALL,M. 0.. EOK, D. and BACHARACH, A. D. E. (1969). Biosynthesis and assembly of the rod outer segment membrane system. Formation and fate of visual pigment in the frog retina. J. molec. Biof. 4!$387-406. G. (1967). Appearance of hydrolase rich granules in HXRSCHHORN, R., HIRXXHORN,K. and WEISSMANN, human lymphocytes induced by phytohemagglutinin and antigens. Blood 30,84-102. KITO, Y., SUSUKI,T., A~UMA,M. and SEKOOVTI, Y. (1968). Absorption spectrum of rhodopsin denatured with acid. Nature, Land. 218,955-956. MARQULL,J. (1970). Acid phosphatase activity in the retinal pigment epithelium. Vision Res. 10, 821-824. SHICHI,H. (1969). Microsomal electron transfer system of bovine retinal pigment epithelium. Exprl Eye Res. 8, 60-68. SHICHI,H. and HACKETT,D. P. (1962). Studies on the b-type cytochromes from mung bean seedlings. II. Some. properties of cytochromes b-555 and b-561. J. Biol. Chem. 237,2959-2964. E, F. and STONE,A. L. (1969). Biochemistry of visual pigments. I. PurificaSHICHI,H., LEWIS,M. S., IRREV~RR tion and properties of bovine rhodopsin. J. Biol. Chem. 244.529-536. YOUNG R. W. and BOK, D. (1969). Participation of the retinal pigment epithelium in the rod outer segment renewal process. J. Cell Biol. 42, 392-403. YOUNO, R. W. and DROZ, B. (1968). The renewal of protein in retinal rods and cones. J. Cell Biol. 39, 169-184.