Exp. Eye Res. (1977) 24, 571-580
Rhodopsin Regeneration in Rod Outer Segments: Utilization 11-cis Retinal and Retinol C. D. B.
of
BRIDGES
Department of Ophthalmology, New York University New York, 2V.Y. 10016, U.S.A.
Medical
Center,
(Received17 September1976, New York) The utilization of aqueous colloidal dispersions of 11-cis retinol, retinal and their 9-&s isomers has been investigated in fragmented and intact frog rod outer segments, the latter while still attached to the retina. The fragments efficiently utilize 75SOo/, of added I I-cis retinal for rhodopsin regeneration, but this proportion drops to about 2% when the plasma membrane is left intact. The corresponding figures for ll-cis retinol are 25 and 0.6% respectively, the former with a NADP+ supplement (NAD+ is less efficacious), the latter with endogenous cofactor. These results suggest that the plasma membrane is an effective barrier to these vitamin A compounds TG t,he form administered. Since the plasma membrane is impermeable to NAD+ and NADP+. the ROS do not lose these cofactors unless fragmented:nor can they take them up if they’are added to the external medium. Formation of &orhodopsin is obser;ed if the 11-c& isom”ers are replaced by 9-&s. dctivity of the 11-cis retinol oxidase is reduced or abolished by washing the ROS fragments (water, 0.64% iVaC1 or 5 mxx-EDT,4), storage at -20” or in the presence of 0.1% Triton X-lOI?. These procedures do not affect all-trams retinal reduction, so it is concluded that the oxidase is a separate entity. Reduction of all-tmns retinal can be linked with oxidation of 11-cis retinol tlqrough the common cofactor NADP(H), so that a coupled cofa,ctor recycling system may exist during light-adaptation in vivo, where bleaching and regeneration occur simultaneously. Key zoords : rhodopsin ; regeneration; rod outer segments ; plasma membrane ; retinol ; retinal; isomers : oxidoreductases ; coupled oxidation-reduction.
1. Introduction The means by which visual pigments are regenerated in the living eye are still not clear; although it has been suggested recently (Bridges, 1976a) that rod outer segments (ROS) enzymatically convert to the ll-cis configuration all-trans retinyl ester transported from the pigment epithelium (RPE). Although both alcohol and aldehyde have also been considered aspossiblecandidat’es,at the present time there is no compelling evidence in favor of a particular derivative of vitamin A as the form isomerized (E’utterman, 1974; Shichi and Somers, 1974; see also Wald, 1968 and Bridges, 1970). Hence, in attempting Do elucidate this system it is important to know which 11-&s compounds can be utilized by the ROS and how efficiently this can be accomplished. In the present study the regeneration of rhodopsin was investigated in bleached preparations of fragmented and intact frog ROS in the presenceof various amounts of 9- and ll-ci.s retinol and retinal as aqueous colloidal dispersions. The results suggestthat the ROS plasma membrane is an effective barrier to these compounds in the form administered, although much mare efficient regeneration occurs when the ROS are thoroughly fragmented. The relatively labile enzyme that oxidizes 11-c& ret.inol to 11-cis retinal appears to be distinct from that which reduces all-trans retinal releasedon bleaching. Since both function with iYADP(H); the two processescan be linked to form a coupled GofaCtorrecycling system. This could happen during light-adaptation in viva, where bleaching and regeneration occur side by side. 571
572
C. D.
B. BRIDGES
2. Materials For the most part, the methods elsewhere (Bridges, 1976a).
and Methods
used in the present paper have been described
in detail
Animals Ra+aa catesbeiana (Mogul Ed and Connecticut Valley Biological), were dark-adapted overnight in incubators at 25”.
4-6 in. in body length,
Tissues
Bfter decapitation, the eyes were enucleated, their front halves and lenses removed, and the retinas dissected complete and free from adhering RPE in Ringer solution (composition as previously listed, except that 10 mnf-Trisacetate was used instead of Tris-HCl: at the pH customarily used, 7.4, no difference between these buffers could be detected). Whole retinas, with intact ROS still attached, were individually bleached and incubated in 1 ml Ringer. Suspensions of ROS fragments in Ringer were prepared as previously described according to the method of Papermaster and Dreyer (1974). Retina.1 and retirkol isomers 11-cis Retinal was separated on PPorasil (Waters) by hplc of an ethanolic photoisomerate of all-tmns retinal (Eastman Kodak). The 9-cis isomer was purchased from Eastman Kodak and the corresponding alcohols obtained by NaBH, reduction of the aldehydes in ethanol. These compounds were dissolved in etha.nol and 2%30~1 were mixed with each ml of incubation mixture. Assay of vitamin
A com~ourzds and photopigment
Retina.1 was measured by a scaled-down version of the calorimetric method of Futterman and Saslaw (1961) and retinol by the Carr-Price reaction as used by Bridges (1975). Rhodopsin and isorhodopsin were extracted by syringing the pelleted ROS or retinas with 0.7-1.0 ml volumes of 2% digitonin (soluble product prepared according to Bridges, 1977), leaving overnight at 4”C, centrifuging and measuring the difference spectrum of the supernatent in the presence of 10 mlm-hydroxylamine. The extraction was repeated twice more. A molar extinction coefficient of 42 000 at 502 nm was used for frog rhodopsin (e.g. Bridges, 1971) and 43 000 at 486 nm for isorhodopsin (Hubbard, Brown and Bownds, 1971). Bleaching,
regeneration
and
retinal reduction
Bfter retention of the contralateral retina or withdrawal of several 1 ml aliquots of suspension as unbleached controls, samples were bleached for 1 hr by exposure to light filtered through a yellow Corning 3-72. All reactions were at 25°C. After incubation, regeneration was stopped by addition of 5 volumes of O-1 ;M-hydroxylamine (pH 7), while reduction of exogenous or endogenous retinal was brought to an end by addition of I volume of 5% ZnSO, as described by Futterman and Saslaw (1961).
3. Results Regeneration
from 11-eis
retinal
The ROS removed from the sucrose gradients and dispersed into Ringer through a 22 ga. needle were in a very fragmented condition. In general, regeneration when
RHODOPSIN
573
REGENERATION
bleached suspensions of this material were mixed with 11-cis retinal was highly efficient, amounting over the 2.5 hr incubation period to 75% for 1 mol equivalent of opsin (6-20 nmol/ml) and lOOo/o if the amount of 11-cis retinal was tripled. Similar results were obtained with the 9-&s isomer. In t#he presence of a fourfold excess of 11-&s retinal, pH had little effect between 5-O and 7.5, but there was some reduction of regeneration above this value. &4t pH 9.0, for example, the amount of rhodopsin regenerated over a 2.5 hr incubation was only 55% of that at pH 7.4. Some of this redudion could be due to the formation of Schiff bases with the excess of -NH, groups available for combination under alkaline conditions. As shown in Fig. 1, relatively massive amounts of 11-&s retinal must be applied to the surface of the intact ROS still attached to the retina in order to obtain significant 1
+---e+?~llO I I- cis retinal,
FIG. 3. Regeneration of rhodopsin from rod outer segments. Note break in abscissa
’ ’
120
molar
excess
Il.& retinal in bleached intact (0) and fragmented between 20 and llO-fold molar excess.
(0)
penetration of the plasma membrane (cf. Pepperberg, Lurie, Brown and Dowling, 1976, who used skate retinas and concentrations of 1.5-15.5~mol/ml, the latter apparently giving 56% regeneration). Some variation in the amount of regeneration was observed with the frog retinas used in the present study, a circumstance possibly related to the variable leakiness of different preparations, as noted when isolated whole retinas are treated with di-dansyl cystine (Yoshikami, Robinson and Hagins, 1974). An approximately 120-fold excess (1000 nmol/ml) of 11-c& retinal was needed to regenerate about 80% of the opsin. On average, it appeared that only 2% (O-6-3.2%) of the ll-cis retinal succeeded in reaching the chromophore site of the bleached opsin in the ROS interior when whole retinas were used. In comparison, the highest observed in ROS fragments was about SO%, under conditions where opsin was present in 2.5-fold excess (13 nmol/ml) over 11-&s retinal. As noted above, however, the utilization is almost asshigh (75%) when opsin and retinal are present in equimolar amounts. Ei’eyenera,tion from 1 l-cis retirwl Bleached ROB fragments regenerate very little from ll-cis retinol unlessthey have been supplemented with NAD+ or NADP+ (see also Bridges, 1976a). The optimum
574
C. D.
B. BRIDGES
pH of the overall reaction is 65-7.5. Unlike the situation with ll-cis retinal, regeneration from the alcohol is markedly reduced under acid conditions, where at pH 5.2 it is only one third of that at pH 7.4. A preference for NADP+ (cf. Bridges, 19’76a) is olear in Table I, where 86% regeneration was observed with NADPf (nos 9 and 10) compared with 50% for NADf (nos ‘7 and 8). Regeneration amounted to about 8% in the absence of cofactor (nos 5 and 6), and only 5% if the ll-cis retinol was omitted (nos 3 and 4). TABLE
Regeneration of rhodopsin
and iso~h&pt&
Preparation-t
I
in bleached suspensions nmol photopigment
Cofactorf
of ROXfragments”
Percent regeneration§
__I_1 2 3 4 5 6 7 8 9 IO 11 12 13 14 15 16 17 18 19 20
unbleached
-
bleached? Il-cis
retinol NAD+ XADP
9-&s retinol NAD+ NADP+ 1 I-&
retinal
9.cis retinal
b
IO.57 9.87 0.47 0.52 0.69 0.79 5.07 4.42 8.42 7.89 1.44 1.29 2.22 2-39 6.43 6.31 9.45 9.59 10.04 IO.23
(111.0) (103,7) 4.9 5.5 7.3 8.3 53.3 464 585 82.9 15.1 13.6 23.3 25.1 67.5 66.3 99.3 100.7 105.5 107.5
* ROS fragments from 34 retinas dispersed after separation on sucrose gradient through 22ga needle into 26 ml Ringer, pH 7.4. -t 1 ml of bleached suspension added to 100 nmol vitamin A compound in 25 pl ethanol. $200 nmol added in 0.2 ml Ringer. 5 Expressed as amount of rhodopsin or isorhodopsin regenerated in 2.5 hr in terms of that obtained with 11-eis retinal (nos 17 and 18). 7 Bleached suspension incubated for 2.5 hr without aclditives.
In the bullfrogs used for the present experiments some variability has been noted over the year, when it was observed that regeneration in the presence of NADf appeared to increase relative to NADP+ in late winter and early spring.* The enzyme converting 11-c& retinol to its aldehyde can also use the 9-&s isomer as a substrate. As shown in Table I, regeneration of isorhodopsin occurs if 9-&s retinol is substituted for 11-&s. In the presence of NADPf this amounted to 67% (nos 15 and 16), but was much lower when NAD+ was the cofactor (nos 13 and 14). Regeneration from 11-&s r&no1 was reduced by as much as 40-60% by various procedures that scarcely affected regeneration from 11-&s retinal. These included washing with water, 0.64% NaCl and 5 rnM-EDTA. Storage of the suspensionfor a week at -20” reduced the observed regeneration by almost two-thirds. The process * See note
added
in proof,
p. 578.
RHODOPSIN
--_ .740
REGEXERATION
was found to be particularly sensitive to Triton X-100. * Normally, retinal or retinol were added as concentrated solutions in small volumes of absolute ethanol. However, if 5% Triton X-100 was present in the ethanol carrier, so that the final concentration of detergent in the medium was 0.13%, then regeneration from ll-cis retinol plus NADP+ was reduced from 90% to nearly zero, as shown in the upper half of Fig. 2, r
o
I 0
I
I IO
I
I 20
I
I
Time (min)
FIG. 2. (a) Oxidation: regeneration of rhodopsin in 1 ml suspensions of bleached fragmented ROS (S-9 nmol opsin) from 11&s retinal, 60 nmol (blocks B) and II-& retinal, 100 nmol, plus xADP+, 200 nmol (blocks A). The hatched areas represent regeneration (1) after I.5 hr preincubation in Ringer containing 0.1% Triton X-100, (a) ROS in Ringer, but 5% Triton X-100 dissolved in the ethanol carrier. (b) Reduction of all-t&u retinal to retinol in 1 ml suspensions of fragmented ROS (S-9 nmol opsin) in the presence of 200 nmol NADH or NADPH. 0, controls in Ringer; 0, after 1.5 hr preincubation in Ringer containing 0.1% Triton X100.
block 2A. Regeneration from ll-cis retinal was only moderately affected (block 2B). Complete abolition of regeneration from II-&s retinal, as well as 11-cis retinol, was observed if the ROS suspension was preincubated in Ringer containing O*lo/o Triton X-100, as shown in blocks 1A and IB. On the other hand, this drastic treatment had little effect on the. reduction of all-trans retinal in the presence of NADH or NADPH. In bovine ROS the latter is strongly preferred as cofactor (Putterman, 1963), and the results in Fig. 2 show that this is also applicable to the frog. Since the reductase is also resistant to the various washing and storage procedures that impaired ll-cis retinol oxidase activity, it appears that the two enzymes are distinct entities. In the living eye, reduction and oxidation take place simultaneously under conditions where a balance between bleaching and regeneration is established during light-adaptation. In suspensions of fragmented ROS it was possible to link the reduction of all-trans retinal with the oxidation of ll-cis retinol through the common cofactor NADP(H), illustrating the feasibility of a coupled cofactor recycling system in vivo. As shown by AA in Pig. 3, bleached suspensions containing endogenous * See note added
in proof,
p. 578.
36
C. D.
B. BRIDGES
alMans retinal regenerate virtually no photopigment. Regeneration is increased slightly in the presence of 114s retinol (no cofactors, block B), but rises to over 80% if the medium is supplemented wit,h NADP+: as expected from the results described above and in Table I. However, significant regeneration is also observed in the presence of 11-cis retinol and the reduced coenzymes: the amount being much great’er ioo-----
NADP+
0
3. X,eduction of all-lrans retinal coupled with oxidation of Il.cis retinol via common cofactors (100 nmol) in bleached suspensions of fragmented ROS (15 nmol opsin). AA, no additives; A, all-traw retinal (75 nmol); B, Il.& retinol (100 nmol); C, all-trams retinal plus 11-&s retinal. FIG.
with NADPH than NADH (blocks B). Clearly, this must be due to the formation of NADP+ (and NAD+) by the enzymatic conversion of endogenous all-tmns retinal to retinal, a reduction not normally observed in such suspensions because the ROS lose their original complement of XADP(H) during fragmentation (see Bridges, 1962 and below). This conclusion is confirmed if; in addition to 11-c& retinol and NADPH, a supplement of exogenous all-trans retinal is added to the medium. This is illustrated
2 50 .-s ‘0 tc P =
FIG. 1. Regeneration no cofactor; B; NAD+
25
of rhodopsin (200 nmol);
from II&S retinol added to intact C, NADPf (200 nmol). Unsupplemented
ROS
(8-13 control
nmol opsin). A, on extreme left.
by block C (NADPH): wh ere the amount regenerated approaches that found with NADP+. As shown by blocks A, there is no increase of regeneration over the control level when all-trans retinal is added, either by itself or with cofactor. As noted above, intact ROX, like mit’ochondria, retain endogenous NADP+ or NAD+, so that regeneration proceeds in isolated retinas on addition of ll-cis retinol
RHODOPSIN
REGENERATIOS
.577
alone. This is demonstrated in Fig. 4, blocks A. Because of the impermeability of the ROS -@ma membrane, it would be expected that exogenous NADP+ (or NAD’) would have little effect on the amounts regenerated. Blocks I3 and C codhn that this is the case. In parallel with the aldehyde, regeneration from II-&s retinol was markedly less when it was added to intact ROS. In fragmented material, the highest observed u.tilization of ll-cis retinol was 25% under conditions where 95% regeneration was obtained when 50 nmol of ll-cis retinol and 100 nmol NADPy were added to about 13 nmol opsin. In Fig. 4; which refers to intact ROS? this is reduced to an average of 0.6% (0.2%1.2%), so that even when massive concentrations of 2000 nmol/ml were added (8-13 nmol opsin/retina), regeneration was boosted to a level no higher than .SO%. 4. Discussion The present work confirms previous observations showing that purified frog ROS rhodopsin when provided with 11-cis retinol and NADP+ (Bridges, 1976a). Isorhodopsin was formed if 9-&s was substituted for 11-&s. The failure by Lion, Rotmans, Daemen and Bonting (1975) to demonstrate the oxidation reaction in bovine material is possibly attributable to their use of preparative methods showu here to be deleterious, viz. washing the ROS several times with distilled water and Jtoring them for unspecified periods of time. The sensitivity of this oxidase when exposed to 0.1% Triton X-100 distinguishes it from the 11-&s retinal oxido-reductase of RPE, which in bovine material is relatively resistant to treatment with ten Dimes this concent,ration (Lion, et al., 1975). In any event, contamination from this source is considered unlikely owing to the care with which the retinas were isolated initially, not a difficult operation in R. catesbeiczna, where the retina, usually floated clean and free within the divided eyecup, secured only by its optic nerve. The involvement of alcohol dehydrogenase may also be ruled out. Unlike liver alcohol dehydrogenase, the retinal enzyme is unreactive with retinol (Koen and Shaw: 1966; Watkins and Tephly, 197 1). The ability of ROS to regenerate visual pigment from 11-cis retinol accounts for the coasumption of stored ll-cis retinyl ester during light-adaptation (see Bridges, 1976a) and the utility for rhodopsin biosynthesis of the small supply of 11-&s retinol in dark-adapted ROS (Bridges, 1976b). Further, it must be concluded that an isomerase converting retinol or its ester precursor from all-trans to 11-&s cannot he eliminated from the visual cycle. The ll-cis retinol oxidase is clearly distinct from the well-studied ROS retinal reductase, the difference being most notable in its loss of activity on storage and treatment with low concentrations of Triton X-100. Moreover, it has been shown that the reductase is highly specific for the all-trans isomer and shows little activity with 11-&s as substrate (in bovine material; Lion et al., 1975).* Experiments exemplified in Fig. 3 demonstrate the possibility that during lightadaptation reduction of all-tyans retinal from rhodopsin bleaching is coupled with oxidation of 11-cis retinol for regeneration through the common cofactor NADP( Hf. Isolated frog retinas or carefully prepared, unwashed ROS are capable of converting nearly all of the all-trans retinal released on bleaching into all-frans retinol (7117ald, 1935; Bridges, X962; Baumann, 1967). In darkness, therefore, the system may be primed in rea,diness for light-adaptation with NADPH generatted win the pentose regenerate
* See note added
in proof,
p. 578.
C. D.
578
B. BRIDGES
cycle (Futterman; Hendrickson, Bishop, Rollins and Vacano, 1970); as summarized in Fig. 5. The present work emphasizes the problem of getting 11-&s retinol and retinal through the ROS plasma membrane. At best utilization of the aldehyde for regeneration drops from 80% in fragmented ROS to about, 2% in the intact organelles. The possibility of competition from other binding sites on the surface of the whole retina is unlikely to be significant, owing to the very high affinity of opsin for ll-cis retinal and the favorable positioning of the ROS that ensures maximal contact area with the surrounding medium. Similarly, 25% of added 11-cis retinol can be converted to rhodopsin in the fragmented material (NADPf supplement), but less than 1% in the whole preparation, where it is assumed that supplies of endogenous cofactor are not limiting. In the latter instance, it was found that only about half of the opsin regenerated with ll-cis retinol in 200-fold molar excess, almost 100 times the amount of vitamin A available in the frog RPE after intensive light-adaptation in vivo. Rhodopsin A
w Phosphqluconote pathway
All-trms retinal -
Reductase
Al I - frans retinoi
T NAD/pH 4
il-;jsS
IV&P+ /
Ii-cis
retinal
retinol
FIG.
5.
Poor utilization of the 11-cis alcohol, even in the ROS fragments, could be due partly to lossesfrom isomerization to all-trans (cf. Stainer and Nlurray, 1960) and autoxidation, a processthat is easily detectable within minutes of dispersing retinol in an aqueous medium (Fisher; Lichti and Lucy, 1972). Under these conditions, retinol apparently forms micelles (Radda and Smith, 1970) that are capable of interacting with erythrocyte stroma and phospholipid vesicles. Recently, however, the existence of low molecular-weight proteins that bind ligands such as retinol has been established in serum and other tissues, including retina (e.g. Kanai, Raz and Goodman, 1968; Bashor, Toft and Chytil, 1973; Gambhir and Ahluwalia: 1974; Ong and Chytil, 1974, 1975; Wiggert: Bergsma and Chader, 1976; Futterman, Saari and Swanson, 1976). These compoundsprobably act as protective agents in the intraand extra-cellular transport of retinol (cf. Futterman and Heller, 1972) as well as providing the means for its “targeted” delivery. Specific binding of plasma holo-retinol-binding protein to pigment epithelium cells has been demonstrated (Heller, 1975) and implicated in the uptake of retinol by the RPE (Maraini and Gozzoli, 1975). Thus the aqueous colloidal dispersions used here are probably not the most efficient way to present retinol (and possibly retinal) to intact cells. Plasma retinol-binding protein normally carries retinol and does not appear to bind to ROS (Bok and Heller, 1976), so it is likely that another carrier protein, perhaps formed by the RPE, would be required if retinol or a derivative such as the palmitate is taken up by the ROS during light- and dark-adaptation. Note added in proof.
Recent experiments have shown that frog ROS reduce all-trans reti.nal faster than the ll-cis isomer. There is now some evidence for the existence of two oxidases, using NAD+ and NADP+ respectively. The activity of the former is enhanced by 1.01 ‘A Triton X-100, but that of the latter is depressed.
RHODOPSIN
REGESERATIOX
579
ACKNOWLEDGMERT The technical assistance of Nr Fouad Farag is acknowledged. This work was supported by PHS Grant No. EY 00461 from the National
Eye Institute.
REFERENCES Bashor. 41. M.. Toft, D. 0. and Chytil, F. (1973). In vitro binding of retinol to rat-tissue components. Proc. Nat. Acad. Sci. U.S.A. 70, 3483-7. Baumann, Chr. (1967). Sehpurpurbleichung und Stabchenfunktion in der isolierten Froschnetzhaut I. Die Sehpurpurbleiohung. Pftigers Archiv. 298,44-60. Bok, D. and Heller, J. (1976). Transport of retinol from blood to retina: an autoradiographic study of the pigment epithelial cell surface receptor for plasma retinol-binding protein. Exp. Eye Res. 22, 395402. Bridges, C. D. B. (1962). Studies on the flash phot.olysis of visual pigments. 4. Dark reactions following flash irradiation of frog rhodopsin in suspensions of isolated photoreceptors. Vision Res. 2, 215-32. Bridges, C. D. B. (1970). Biochemistry of vision. In Biochemistry of the Eye (Ed. Graymore, C. N.). Pp. 563-644. Academic Press, New York. Bridges, C. D. B. (1971). The molar absorbance coefficient of rhodopsin. Vision Res. 11, 841-8. Bridges, C. D. B. (1975). Storage, distribution and utilization of vitamins A in the eyes of adult’ amphibians and their tadpoles. vision Res. 15, 1311-23. Bridges, C. D. B. (1976a). Vitamin A and the role of the pigment epithelium during bleaching and regeneration of rhodopsin in the frog eye. Exp. Eye Res. 22,435-55. Bridges, C. D. B. (197613). 11-cis vitamin A in dark-adapted rod outer segments is a probable source of prosthetic groups in rhodopsin biosynthesis. Nature, Lord. 259, 247-S. Bridges, C. D. B. (1977). A method for preparing stable digitonin solutions for visual pigment extraction. Vision Res., 17, 301-2. Fisher, D., Licht.i, F. U. and Lucy, J. A. (1972). Environmental effects on the autoxidation of retinol. Biochem. J. 130, 259-70. Futterman, S. (1963). Metabolism of the retina. 111. The role of reduced triphosphopyridine nucleotide in the visual cycle. J. Biol. Chem. 238, 1145-50. Futterman, S. (1974). Recent studies on a possible mechanism for visual pigment regeneration. Exp. Eye Res. 18, 89-96. Futterman, S. and Heller, J. (1972). The enhancement of fluorescence and the decreased susceptibility to enzymatic oxidation of retinol complexed with bovine serum albumin./?-lactoglobulin, and the retinol-binding protein of human plasma. d. Biol. Chem. 247, 5168-72. Futterman, S., Hendrickson, A., Bishop, P. E., Rollins, M. H., and Vacano, E. (1970). Metabolism of glucose and reduction of retinaldehyde in retinal photoreceptors. J. NeurochenL. 17,149~56. Futterman, S., Saari, J. C. and Swanson: D. E. (1976). Retinol and retinoic acid-binding proteins in bovine retina: aspects of binding specificity. Exp. Eye Res. 22, 419-24. Futterman, S. and Saslaw, L. D. (1961). The estimation of vitamin A aldehyde with thiobarbituric acid. J. Biol. Chem. 236, 1652-7. Gambhir, K. K. and Ahluwalia, B. S. (1974). A smaller molecular weight retinol binding protein in rat testis seminiferous tubules. Biochem. Biophys. Res. Commun. 61, 551-8. Heller, J. (1975). Interactions of plasma retinol-binding protein with its receptor. J. Biol. Chews. 250,3613-g. Hubbard. R., Brown, P. K. and Bownds, D. (1971). Methodology of vitamin A and visual pigments. In Methods in Enzymology 18, Vitawks and Coenzymes (Eds McCormick, D. B. and Wright, L. D.). Pp. 615-53. Academic Press, Kew York. Kanai. M., Raz, A. and Goodman, De W. S. (1968). Retinol-binding protein: the transport protein for vitamin A in human plasma. J. Clin. Invest. 47, 202544. Koen, A. L. and Shaw, C. R. (1966). Retinol and alcohol dehydrogenases in retina and liver. B&&em. Bi0phy.s. Acta 128, 48-54. Lion, F., Rotmans, J. P., Daemen, F. J. M. and Bonting, S. L. (1975). Stereospecificity of ocular retinol dehydrogenases and the visual cycle. Biochim. Biophys. Acta 384, 283-92. Maraini, G. and Gozzoli, F. (1975). Binding of retinol to isolated retina1 pigment epithelium in. the presence and absence of retinoI-binding protein. Invest. Ophthalmol. 14, 785-7.
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D. E. and Chytil, F. (1974). Multiple retinol binding proteins in rabbit lung. Biodenz. Biophys. Res. Commun. 59, 221-9. Ong, D. E. and Chytil, F. (1975). Specificity of cellular retinol-binding protein for compounds with vitamin A activity. Nature, Lord. 259, 74-5. Papermaster, D. S. and Dreyer, W. J. (1974). Rhodopsin content in the outer segment membranes of bovine and frog ret,inal rods. Biochemistry 13, 243844. Pepperberg, D. R., Lurie, M., Brown, P. K. and Dowling, J. E. (1976). Visual adaptation: effects of externally applied retinal on the light-adapted, isolated skate retina. Science 191, 394-6. Radda, G. K. and Smith, D. S. (1970). Retinol: a fluorescent probe for membrane lipids. FEBS Lett. 9, 287-9. Shichi, H. and Somers, R. L. (1974). Possible involvement of retinylidenephospholipid in photoisomerization of all-tvans retinal to ll-cis retinal. J. Biol. Chem. 249, 6570-77. Stainer, D. W. and Murray, T. K. (1960). Isomerization of vitamin A by tissue homogenates. Canad. J. Biochem. Physiol. 38, 1467-70. Wald, G. (1935). Carotenoids and the visual cycle. J. Gen. Physiol. 19, 351-71. Wald, G. (1968). The molecular basis of visual excitation. Nature, Lond. 219, 800-807. Watkins: W. D. and Tephly, T. R. (1971). Studies on the properties of retinal alcohol dehydrogena,se from the rat. J. Neuroch,em. 18, 2397-406. Wiggert, B. O., Bergsma, D. R. and Chader, G. J. (1976). Retinol receptors of the retina and pigment epithelium: further charact’eriza,tion and species variation. Exp. Eye Res. 22,411-18. Yoshikami, S., Robinson, W. E. and Hagins, W. A. (1974). Topology of the outer segment membranes of retinal rods and cones revealed by a fluorescent, probe. Science 185, 1176-9.
Rhodopsin Regeneration in Rod Outer Segments: Utilization 11-cis Retinal and Retinol C. D. B.
of
BRIDGES
Department of Ophthalmology, New York University New York, 2V.Y. 10016, U.S.A.
Medical
Center,
(Received17 September1976, New York) The utilization of aqueous colloidal dispersions of 11-cis retinol, retinal and their 9-&s isomers has been investigated in fragmented and intact frog rod outer segments, the latter while still attached to the retina. The fragments efficiently utilize 75SOo/, of added I I-cis retinal for rhodopsin regeneration, but this proportion drops to about 2% when the plasma membrane is left intact. The corresponding figures for ll-cis retinol are 25 and 0.6% respectively, the former with a NADP+ supplement (NAD+ is less efficacious), the latter with endogenous cofactor. These results suggest that the plasma membrane is an effective barrier to these vitamin A compounds TG t,he form administered. Since the plasma membrane is impermeable to NAD+ and NADP+. the ROS do not lose these cofactors unless fragmented:nor can they take them up if they’are added to the external medium. Formation of &orhodopsin is obser;ed if the 11-c& isom”ers are replaced by 9-&s. dctivity of the 11-cis retinol oxidase is reduced or abolished by washing the ROS fragments (water, 0.64% iVaC1 or 5 mxx-EDT,4), storage at -20” or in the presence of 0.1% Triton X-lOI?. These procedures do not affect all-trams retinal reduction, so it is concluded that the oxidase is a separate entity. Reduction of all-tmns retinal can be linked with oxidation of 11-cis retinol tlqrough the common cofactor NADP(H), so that a coupled cofa,ctor recycling system may exist during light-adaptation in vivo, where bleaching and regeneration occur simultaneously. Key zoords : rhodopsin ; regeneration; rod outer segments ; plasma membrane ; retinol ; retinal; isomers : oxidoreductases ; coupled oxidation-reduction.
1. Introduction The means by which visual pigments are regenerated in the living eye are still not clear; although it has been suggested recently (Bridges, 1976a) that rod outer segments (ROS) enzymatically convert to the ll-cis configuration all-trans retinyl ester transported from the pigment epithelium (RPE). Although both alcohol and aldehyde have also been considered aspossiblecandidat’es,at the present time there is no compelling evidence in favor of a particular derivative of vitamin A as the form isomerized (E’utterman, 1974; Shichi and Somers, 1974; see also Wald, 1968 and Bridges, 1970). Hence, in attempting Do elucidate this system it is important to know which 11-&s compounds can be utilized by the ROS and how efficiently this can be accomplished. In the present study the regeneration of rhodopsin was investigated in bleached preparations of fragmented and intact frog ROS in the presenceof various amounts of 9- and ll-ci.s retinol and retinal as aqueous colloidal dispersions. The results suggestthat the ROS plasma membrane is an effective barrier to these compounds in the form administered, although much mare efficient regeneration occurs when the ROS are thoroughly fragmented. The relatively labile enzyme that oxidizes 11-c& ret.inol to 11-cis retinal appears to be distinct from that which reduces all-trans retinal releasedon bleaching. Since both function with iYADP(H); the two processescan be linked to form a coupled GofaCtorrecycling system. This could happen during light-adaptation in viva, where bleaching and regeneration occur side by side. 571
572
C. D.
B. BRIDGES
2. Materials For the most part, the methods elsewhere (Bridges, 1976a).
and Methods
used in the present paper have been described
in detail
Animals Ra+aa catesbeiana (Mogul Ed and Connecticut Valley Biological), were dark-adapted overnight in incubators at 25”.
4-6 in. in body length,
Tissues
Bfter decapitation, the eyes were enucleated, their front halves and lenses removed, and the retinas dissected complete and free from adhering RPE in Ringer solution (composition as previously listed, except that 10 mnf-Trisacetate was used instead of Tris-HCl: at the pH customarily used, 7.4, no difference between these buffers could be detected). Whole retinas, with intact ROS still attached, were individually bleached and incubated in 1 ml Ringer. Suspensions of ROS fragments in Ringer were prepared as previously described according to the method of Papermaster and Dreyer (1974). Retina.1 and retirkol isomers 11-cis Retinal was separated on PPorasil (Waters) by hplc of an ethanolic photoisomerate of all-tmns retinal (Eastman Kodak). The 9-cis isomer was purchased from Eastman Kodak and the corresponding alcohols obtained by NaBH, reduction of the aldehydes in ethanol. These compounds were dissolved in etha.nol and 2%30~1 were mixed with each ml of incubation mixture. Assay of vitamin
A com~ourzds and photopigment
Retina.1 was measured by a scaled-down version of the calorimetric method of Futterman and Saslaw (1961) and retinol by the Carr-Price reaction as used by Bridges (1975). Rhodopsin and isorhodopsin were extracted by syringing the pelleted ROS or retinas with 0.7-1.0 ml volumes of 2% digitonin (soluble product prepared according to Bridges, 1977), leaving overnight at 4”C, centrifuging and measuring the difference spectrum of the supernatent in the presence of 10 mlm-hydroxylamine. The extraction was repeated twice more. A molar extinction coefficient of 42 000 at 502 nm was used for frog rhodopsin (e.g. Bridges, 1971) and 43 000 at 486 nm for isorhodopsin (Hubbard, Brown and Bownds, 1971). Bleaching,
regeneration
and
retinal reduction
Bfter retention of the contralateral retina or withdrawal of several 1 ml aliquots of suspension as unbleached controls, samples were bleached for 1 hr by exposure to light filtered through a yellow Corning 3-72. All reactions were at 25°C. After incubation, regeneration was stopped by addition of 5 volumes of O-1 ;M-hydroxylamine (pH 7), while reduction of exogenous or endogenous retinal was brought to an end by addition of I volume of 5% ZnSO, as described by Futterman and Saslaw (1961).
3. Results Regeneration
from 11-eis
retinal
The ROS removed from the sucrose gradients and dispersed into Ringer through a 22 ga. needle were in a very fragmented condition. In general, regeneration when
RHODOPSIN
573
REGENERATION
bleached suspensions of this material were mixed with 11-cis retinal was highly efficient, amounting over the 2.5 hr incubation period to 75% for 1 mol equivalent of opsin (6-20 nmol/ml) and lOOo/o if the amount of 11-cis retinal was tripled. Similar results were obtained with the 9-&s isomer. In t#he presence of a fourfold excess of 11-&s retinal, pH had little effect between 5-O and 7.5, but there was some reduction of regeneration above this value. &4t pH 9.0, for example, the amount of rhodopsin regenerated over a 2.5 hr incubation was only 55% of that at pH 7.4. Some of this redudion could be due to the formation of Schiff bases with the excess of -NH, groups available for combination under alkaline conditions. As shown in Fig. 1, relatively massive amounts of 11-&s retinal must be applied to the surface of the intact ROS still attached to the retina in order to obtain significant 1
+---e+?~llO I I- cis retinal,
FIG. 3. Regeneration of rhodopsin from rod outer segments. Note break in abscissa
’ ’
120
molar
excess
Il.& retinal in bleached intact (0) and fragmented between 20 and llO-fold molar excess.
(0)
penetration of the plasma membrane (cf. Pepperberg, Lurie, Brown and Dowling, 1976, who used skate retinas and concentrations of 1.5-15.5~mol/ml, the latter apparently giving 56% regeneration). Some variation in the amount of regeneration was observed with the frog retinas used in the present study, a circumstance possibly related to the variable leakiness of different preparations, as noted when isolated whole retinas are treated with di-dansyl cystine (Yoshikami, Robinson and Hagins, 1974). An approximately 120-fold excess (1000 nmol/ml) of 11-c& retinal was needed to regenerate about 80% of the opsin. On average, it appeared that only 2% (O-6-3.2%) of the ll-cis retinal succeeded in reaching the chromophore site of the bleached opsin in the ROS interior when whole retinas were used. In comparison, the highest observed in ROS fragments was about SO%, under conditions where opsin was present in 2.5-fold excess (13 nmol/ml) over 11-&s retinal. As noted above, however, the utilization is almost asshigh (75%) when opsin and retinal are present in equimolar amounts. Ei’eyenera,tion from 1 l-cis retirwl Bleached ROB fragments regenerate very little from ll-cis retinol unlessthey have been supplemented with NAD+ or NADP+ (see also Bridges, 1976a). The optimum
574
C. D.
B. BRIDGES
pH of the overall reaction is 65-7.5. Unlike the situation with ll-cis retinal, regeneration from the alcohol is markedly reduced under acid conditions, where at pH 5.2 it is only one third of that at pH 7.4. A preference for NADP+ (cf. Bridges, 19’76a) is olear in Table I, where 86% regeneration was observed with NADPf (nos 9 and 10) compared with 50% for NADf (nos ‘7 and 8). Regeneration amounted to about 8% in the absence of cofactor (nos 5 and 6), and only 5% if the ll-cis retinol was omitted (nos 3 and 4). TABLE
Regeneration of rhodopsin
and iso~h&pt&
Preparation-t
I
in bleached suspensions nmol photopigment
Cofactorf
of ROXfragments”
Percent regeneration§
__I_1 2 3 4 5 6 7 8 9 IO 11 12 13 14 15 16 17 18 19 20
unbleached
-
bleached? Il-cis
retinol NAD+ XADP
9-&s retinol NAD+ NADP+ 1 I-&
retinal
9.cis retinal
b
IO.57 9.87 0.47 0.52 0.69 0.79 5.07 4.42 8.42 7.89 1.44 1.29 2.22 2-39 6.43 6.31 9.45 9.59 10.04 IO.23
(111.0) (103,7) 4.9 5.5 7.3 8.3 53.3 464 585 82.9 15.1 13.6 23.3 25.1 67.5 66.3 99.3 100.7 105.5 107.5
* ROS fragments from 34 retinas dispersed after separation on sucrose gradient through 22ga needle into 26 ml Ringer, pH 7.4. -t 1 ml of bleached suspension added to 100 nmol vitamin A compound in 25 pl ethanol. $200 nmol added in 0.2 ml Ringer. 5 Expressed as amount of rhodopsin or isorhodopsin regenerated in 2.5 hr in terms of that obtained with 11-eis retinal (nos 17 and 18). 7 Bleached suspension incubated for 2.5 hr without aclditives.
In the bullfrogs used for the present experiments some variability has been noted over the year, when it was observed that regeneration in the presence of NADf appeared to increase relative to NADP+ in late winter and early spring.* The enzyme converting 11-c& retinol to its aldehyde can also use the 9-&s isomer as a substrate. As shown in Table I, regeneration of isorhodopsin occurs if 9-&s retinol is substituted for 11-&s. In the presence of NADPf this amounted to 67% (nos 15 and 16), but was much lower when NAD+ was the cofactor (nos 13 and 14). Regeneration from 11-&s r&no1 was reduced by as much as 40-60% by various procedures that scarcely affected regeneration from 11-&s retinal. These included washing with water, 0.64% NaCl and 5 rnM-EDTA. Storage of the suspensionfor a week at -20” reduced the observed regeneration by almost two-thirds. The process * See note
added
in proof,
p. 578.
RHODOPSIN
--_ .740
REGEXERATION
was found to be particularly sensitive to Triton X-100. * Normally, retinal or retinol were added as concentrated solutions in small volumes of absolute ethanol. However, if 5% Triton X-100 was present in the ethanol carrier, so that the final concentration of detergent in the medium was 0.13%, then regeneration from ll-cis retinol plus NADP+ was reduced from 90% to nearly zero, as shown in the upper half of Fig. 2, r
o
I 0
I
I IO
I
I 20
I
I
Time (min)
FIG. 2. (a) Oxidation: regeneration of rhodopsin in 1 ml suspensions of bleached fragmented ROS (S-9 nmol opsin) from 11&s retinal, 60 nmol (blocks B) and II-& retinal, 100 nmol, plus xADP+, 200 nmol (blocks A). The hatched areas represent regeneration (1) after I.5 hr preincubation in Ringer containing 0.1% Triton X-100, (a) ROS in Ringer, but 5% Triton X-100 dissolved in the ethanol carrier. (b) Reduction of all-t&u retinal to retinol in 1 ml suspensions of fragmented ROS (S-9 nmol opsin) in the presence of 200 nmol NADH or NADPH. 0, controls in Ringer; 0, after 1.5 hr preincubation in Ringer containing 0.1% Triton X100.
block 2A. Regeneration from ll-cis retinal was only moderately affected (block 2B). Complete abolition of regeneration from II-&s retinal, as well as 11-cis retinol, was observed if the ROS suspension was preincubated in Ringer containing O*lo/o Triton X-100, as shown in blocks 1A and IB. On the other hand, this drastic treatment had little effect on the. reduction of all-trans retinal in the presence of NADH or NADPH. In bovine ROS the latter is strongly preferred as cofactor (Putterman, 1963), and the results in Fig. 2 show that this is also applicable to the frog. Since the reductase is also resistant to the various washing and storage procedures that impaired ll-cis retinol oxidase activity, it appears that the two enzymes are distinct entities. In the living eye, reduction and oxidation take place simultaneously under conditions where a balance between bleaching and regeneration is established during light-adaptation. In suspensions of fragmented ROS it was possible to link the reduction of all-trans retinal with the oxidation of ll-cis retinol through the common cofactor NADP(H), illustrating the feasibility of a coupled cofactor recycling system in vivo. As shown by AA in Pig. 3, bleached suspensions containing endogenous * See note added
in proof,
p. 578.
36
C. D.
B. BRIDGES
alMans retinal regenerate virtually no photopigment. Regeneration is increased slightly in the presence of 114s retinol (no cofactors, block B), but rises to over 80% if the medium is supplemented wit,h NADP+: as expected from the results described above and in Table I. However, significant regeneration is also observed in the presence of 11-cis retinol and the reduced coenzymes: the amount being much great’er ioo-----
NADP+
0
3. X,eduction of all-lrans retinal coupled with oxidation of Il.cis retinol via common cofactors (100 nmol) in bleached suspensions of fragmented ROS (15 nmol opsin). AA, no additives; A, all-traw retinal (75 nmol); B, Il.& retinol (100 nmol); C, all-trams retinal plus 11-&s retinal. FIG.
with NADPH than NADH (blocks B). Clearly, this must be due to the formation of NADP+ (and NAD+) by the enzymatic conversion of endogenous all-tmns retinal to retinal, a reduction not normally observed in such suspensions because the ROS lose their original complement of XADP(H) during fragmentation (see Bridges, 1962 and below). This conclusion is confirmed if; in addition to 11-c& retinol and NADPH, a supplement of exogenous all-trans retinal is added to the medium. This is illustrated
2 50 .-s ‘0 tc P =
FIG. 1. Regeneration no cofactor; B; NAD+
25
of rhodopsin (200 nmol);
from II&S retinol added to intact C, NADPf (200 nmol). Unsupplemented
ROS
(8-13 control
nmol opsin). A, on extreme left.
by block C (NADPH): wh ere the amount regenerated approaches that found with NADP+. As shown by blocks A, there is no increase of regeneration over the control level when all-trans retinal is added, either by itself or with cofactor. As noted above, intact ROX, like mit’ochondria, retain endogenous NADP+ or NAD+, so that regeneration proceeds in isolated retinas on addition of ll-cis retinol
RHODOPSIN
REGENERATIOS
.577
alone. This is demonstrated in Fig. 4, blocks A. Because of the impermeability of the ROS -@ma membrane, it would be expected that exogenous NADP+ (or NAD’) would have little effect on the amounts regenerated. Blocks I3 and C codhn that this is the case. In parallel with the aldehyde, regeneration from II-&s retinol was markedly less when it was added to intact ROS. In fragmented material, the highest observed u.tilization of ll-cis retinol was 25% under conditions where 95% regeneration was obtained when 50 nmol of ll-cis retinol and 100 nmol NADPy were added to about 13 nmol opsin. In Fig. 4; which refers to intact ROS? this is reduced to an average of 0.6% (0.2%1.2%), so that even when massive concentrations of 2000 nmol/ml were added (8-13 nmol opsin/retina), regeneration was boosted to a level no higher than .SO%. 4. Discussion The present work confirms previous observations showing that purified frog ROS rhodopsin when provided with 11-cis retinol and NADP+ (Bridges, 1976a). Isorhodopsin was formed if 9-&s was substituted for 11-&s. The failure by Lion, Rotmans, Daemen and Bonting (1975) to demonstrate the oxidation reaction in bovine material is possibly attributable to their use of preparative methods showu here to be deleterious, viz. washing the ROS several times with distilled water and Jtoring them for unspecified periods of time. The sensitivity of this oxidase when exposed to 0.1% Triton X-100 distinguishes it from the 11-&s retinal oxido-reductase of RPE, which in bovine material is relatively resistant to treatment with ten Dimes this concent,ration (Lion, et al., 1975). In any event, contamination from this source is considered unlikely owing to the care with which the retinas were isolated initially, not a difficult operation in R. catesbeiczna, where the retina, usually floated clean and free within the divided eyecup, secured only by its optic nerve. The involvement of alcohol dehydrogenase may also be ruled out. Unlike liver alcohol dehydrogenase, the retinal enzyme is unreactive with retinol (Koen and Shaw: 1966; Watkins and Tephly, 197 1). The ability of ROS to regenerate visual pigment from 11-cis retinol accounts for the coasumption of stored ll-cis retinyl ester during light-adaptation (see Bridges, 1976a) and the utility for rhodopsin biosynthesis of the small supply of 11-&s retinol in dark-adapted ROS (Bridges, 1976b). Further, it must be concluded that an isomerase converting retinol or its ester precursor from all-trans to 11-&s cannot he eliminated from the visual cycle. The ll-cis retinol oxidase is clearly distinct from the well-studied ROS retinal reductase, the difference being most notable in its loss of activity on storage and treatment with low concentrations of Triton X-100. Moreover, it has been shown that the reductase is highly specific for the all-trans isomer and shows little activity with 11-&s as substrate (in bovine material; Lion et al., 1975).* Experiments exemplified in Fig. 3 demonstrate the possibility that during lightadaptation reduction of all-tyans retinal from rhodopsin bleaching is coupled with oxidation of 11-cis retinol for regeneration through the common cofactor NADP( Hf. Isolated frog retinas or carefully prepared, unwashed ROS are capable of converting nearly all of the all-trans retinal released on bleaching into all-frans retinol (7117ald, 1935; Bridges, X962; Baumann, 1967). In darkness, therefore, the system may be primed in rea,diness for light-adaptation with NADPH generatted win the pentose regenerate
* See note added
in proof,
p. 578.
C. D.
578
B. BRIDGES
cycle (Futterman; Hendrickson, Bishop, Rollins and Vacano, 1970); as summarized in Fig. 5. The present work emphasizes the problem of getting 11-&s retinol and retinal through the ROS plasma membrane. At best utilization of the aldehyde for regeneration drops from 80% in fragmented ROS to about, 2% in the intact organelles. The possibility of competition from other binding sites on the surface of the whole retina is unlikely to be significant, owing to the very high affinity of opsin for ll-cis retinal and the favorable positioning of the ROS that ensures maximal contact area with the surrounding medium. Similarly, 25% of added 11-cis retinol can be converted to rhodopsin in the fragmented material (NADPf supplement), but less than 1% in the whole preparation, where it is assumed that supplies of endogenous cofactor are not limiting. In the latter instance, it was found that only about half of the opsin regenerated with ll-cis retinol in 200-fold molar excess, almost 100 times the amount of vitamin A available in the frog RPE after intensive light-adaptation in vivo. Rhodopsin A
w Phosphqluconote pathway
All-trms retinal -
Reductase
Al I - frans retinoi
T NAD/pH 4
il-;jsS
IV&P+ /
Ii-cis
retinal
retinol
FIG.
5.
Poor utilization of the 11-cis alcohol, even in the ROS fragments, could be due partly to lossesfrom isomerization to all-trans (cf. Stainer and Nlurray, 1960) and autoxidation, a processthat is easily detectable within minutes of dispersing retinol in an aqueous medium (Fisher; Lichti and Lucy, 1972). Under these conditions, retinol apparently forms micelles (Radda and Smith, 1970) that are capable of interacting with erythrocyte stroma and phospholipid vesicles. Recently, however, the existence of low molecular-weight proteins that bind ligands such as retinol has been established in serum and other tissues, including retina (e.g. Kanai, Raz and Goodman, 1968; Bashor, Toft and Chytil, 1973; Gambhir and Ahluwalia: 1974; Ong and Chytil, 1974, 1975; Wiggert: Bergsma and Chader, 1976; Futterman, Saari and Swanson, 1976). These compoundsprobably act as protective agents in the intraand extra-cellular transport of retinol (cf. Futterman and Heller, 1972) as well as providing the means for its “targeted” delivery. Specific binding of plasma holo-retinol-binding protein to pigment epithelium cells has been demonstrated (Heller, 1975) and implicated in the uptake of retinol by the RPE (Maraini and Gozzoli, 1975). Thus the aqueous colloidal dispersions used here are probably not the most efficient way to present retinol (and possibly retinal) to intact cells. Plasma retinol-binding protein normally carries retinol and does not appear to bind to ROS (Bok and Heller, 1976), so it is likely that another carrier protein, perhaps formed by the RPE, would be required if retinol or a derivative such as the palmitate is taken up by the ROS during light- and dark-adaptation. Note added in proof.
Recent experiments have shown that frog ROS reduce all-trans reti.nal faster than the ll-cis isomer. There is now some evidence for the existence of two oxidases, using NAD+ and NADP+ respectively. The activity of the former is enhanced by 1.01 ‘A Triton X-100, but that of the latter is depressed.
RHODOPSIN
REGESERATIOX
579
ACKNOWLEDGMERT The technical assistance of Nr Fouad Farag is acknowledged. This work was supported by PHS Grant No. EY 00461 from the National
Eye Institute.
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D. E. and Chytil, F. (1974). Multiple retinol binding proteins in rabbit lung. Biodenz. Biophys. Res. Commun. 59, 221-9. Ong, D. E. and Chytil, F. (1975). Specificity of cellular retinol-binding protein for compounds with vitamin A activity. Nature, Lord. 259, 74-5. Papermaster, D. S. and Dreyer, W. J. (1974). Rhodopsin content in the outer segment membranes of bovine and frog ret,inal rods. Biochemistry 13, 243844. Pepperberg, D. R., Lurie, M., Brown, P. K. and Dowling, J. E. (1976). Visual adaptation: effects of externally applied retinal on the light-adapted, isolated skate retina. Science 191, 394-6. Radda, G. K. and Smith, D. S. (1970). Retinol: a fluorescent probe for membrane lipids. FEBS Lett. 9, 287-9. Shichi, H. and Somers, R. L. (1974). Possible involvement of retinylidenephospholipid in photoisomerization of all-tvans retinal to ll-cis retinal. J. Biol. Chem. 249, 6570-77. Stainer, D. W. and Murray, T. K. (1960). Isomerization of vitamin A by tissue homogenates. Canad. J. Biochem. Physiol. 38, 1467-70. Wald, G. (1935). Carotenoids and the visual cycle. J. Gen. Physiol. 19, 351-71. Wald, G. (1968). The molecular basis of visual excitation. Nature, Lond. 219, 800-807. Watkins: W. D. and Tephly, T. R. (1971). Studies on the properties of retinal alcohol dehydrogena,se from the rat. J. Neuroch,em. 18, 2397-406. Wiggert, B. O., Bergsma, D. R. and Chader, G. J. (1976). Retinol receptors of the retina and pigment epithelium: further charact’eriza,tion and species variation. Exp. Eye Res. 22,411-18. Yoshikami, S., Robinson, W. E. and Hagins, W. A. (1974). Topology of the outer segment membranes of retinal rods and cones revealed by a fluorescent, probe. Science 185, 1176-9.
