The contribution of a sensitizing pigment to the photosensitivity spectra of fly rhodopsin and metarhodopsin.

Most of the photoreceptors of the fly compound eye have high sensitivity in the ultraviolet (UV) as well as in the visible spectral range. This UV sensitivity arises from a photostable pigment that acts as a sensitizer for rhodopsin. Because the sensitizing pigment cannot be bleached, the classical determination of the photosensitivity spectrum from measurements of the difference spectrum of the pigment cannot be applied. We therefore used a new method to determine the photosensitivity spectra of rhodopsin and metarhodopsin in the UV spectral range. The method is based on the fact that the invertebrate visual pigment is a bistable one, in which rhodopsin and metarhodopsin are photointerconvertible. The pigment changes were measured by a fast electrical potential, called the M potential, which arises from activation of metarhodopsin. We first established the use of the M potential as a reliable measure of the visual pigment changes in the fly. We then calculated the photosensitivity spectrum of rhodopsin and metarhodopsin by using two kinds of experimentally measured spectra: the relaxation and the photoequilibrium spectra. The relaxation spectrum represents the wavelength dependence of the rate of approach of the pigment molecules to photoequilibrium. This spectrum is the weighted sum of the photosensitivity spectra of rhodopsin and metarhodopsin. The photoequilibrium spectrum measures the fraction of metarhodopsin (or rhodopsin) in photoequilibrium which is reached in the steady state for application of various wavelengths of light. By using this method we found that, although the photosensitivity spectra of rhodopsin and metarhodopsin are very different in the visible, they show strict coincidence in the UV region. This observation indicates that the photostable pigment acts as a sensitizer for both rhodopsin as well as metarhodopsin.     

Visual pigment spectra of the comma butterfly,<Emphasis Type="Italic"> Polygonia c-album</Emphasis>, derived from in vivo epi-illumination microspectrophotometry

The visual pigments in the compound eye of the comma butterfly, Polygonia c-album, were investigated in a specially designed epi-illumination microspectrophotometer. Absorption changes due to photochemical conversions of the visual pigments, or due to light-independent visual pigment decay and regeneration, were studied by measuring the eye shine, i.e., the light reflected from the tapetum located in each ommatidium proximal to the visual pigment-bearing rhabdom. The obtained absorbance difference spectra demonstrated the dominant presence of a green visual pigment. The rhodopsin and its metarhodopsin have absorption peak wavelengths at 532 nm and 492 nm, respectively. The metarhodopsin is removed from the rhabdom with a time constant of 15 min and the rhodopsin is regenerated with a time constant of 59 min (room temperature). A UV rhodopsin with metarhodopsin absorbing maximally at 467 nm was revealed, and evidence for a blue rhodopsin was obtained indirectly.     

Dark regeneration of rhodopsin in crayfish photoreceptors

The eyes of crayfish were exposed to lights of known spectral composition, and the course of regeneration was followed in the dark by measuring the content of rhodopsin and metarhodopsin in single rhabdoms isolated at various times after the adaptation, using an assay that is based on the fluorescence of metarhodopsin. Complete recovery requires several days in the dark after intense adaptation to orange light, but requires less than 2 d after blue light exposure. Following an orange light exposure with blue produces recovery kinetics characteristic of the blue light exposure alone. This quickening of recovery occurs whether the receptors are exposed to blue light either immediately or many hours after the original exposure to orange. Conversely, following blue light adaptation with orange leads to slow recovery, which is characteristic of orange alone. Recovery from long-wavelength adaptation is slower principally because many rhabdoms seem to delay the onset of regeneration. We suggest that the regeneration system is itself photosensitive, and after orange light adaptation the supply of active chromophore (presumably 11-cis retinal) limits the rate of recovery. Once started, recovery proceeds slowly and continuously, and the total pigment concentration (rhodopsin plus metarhodopsin) in the rhabdomeric membrane remains approximately constant. Within hours after intense adapting exposures, the rhabdoms become altered in appearance, the surfaces become coated with accessory pigment, and the bands of microvilli are less distinct. These changes persist until recovery of rhodopsin proceeds, which suggests that visual pigment regeneration results from addition of newly synthesized rhodopsin associated with membrane turn-over.     

The visual pigment and visual cycle of the lobster,Homarus

Summary The visual pigment of the American lobster,Homarus americanus, has been studied in individual isolated rhabdoms by microspectrophotometry. Lobster rhodopsin has _max at 515 nm and is converted by light to a stable metarhodopsin with _max at 490 nm. These figures are in good agreement with corresponding values obtained by Wald and Hubbard (1957) in digitonin extracts. Photoregeneration of rhodopsin to metarhodopsin is also observed. The absorbance spectrum of lobster metarhodopsin is invariant with pH in the range 5.4–9, indicating that even after isomerization of the chromophore fromcis totrans, the binding site of the chromophore remains sequestered from the solvent environment. Total axial density of the lobster rhabdom to unpolarized light is about 0.7.As described for several other Crustacea, aldehyde fixation renders the metarhodopsin susceptible to photobleaching, a process that is faster at alkaline than at neutral or acid pH. Small amounts of a photoproduct with _max at 370 nm are occasionally seen. A slower dark bleaching of lobster rhabdoms (_1/2–2 h) also occurs, frequently through intermediates with absorption similar to metarhodopsin.The molar extinction coefficient of metarhodopsin is about 1.2 times greater than that of rhodopsin, each measured at their respective _max. Isomerization of the chromophore fromcis totrans is accompanied by a change in the orientation of the absorption vector of about 3°. The absorption vector of metarhodopsin is either tilted more steeply into the membrane or is less tightly oriented with respect to the microvillar axes.When living lobsters are kept at room temperature, light adaptation does not result in an accumulation of metarhodopsin. At 4 °C, however, the same adapting lights cause a reduction of rhodopsin and an increase in metarhodopsin. There is thus a temperature-sensitive regeneration mechanism that supplements photoregeneration. Following 1 ms, 0.1 joule xenon flashes that convert about 70% of the rhodopsin to metarhodopsin in vivo, dark regeneration occurs in the living eye with half-times of about 25 and 55 min at 22 °C and 15 °C respectively.This work was supported by USPHS research grant EY 00222 to Yale University. S.N.B. was aided by NIH Postdoctoral Fellowship EY 52378.     

Microspectrophotometry of rhodopsin and metarhodopsin in the moth Galleria

Fresh, frozen sections of the photoreceptor layer of the compound eye of the moth Galleria have been examined by microspectrophotometry, using 4 X 8 mum measuring beams that sampled from approximately two to four rhabdoms. The principal visual pigmen: absorbs maximally at 510 nm (P510), and on irradiation is converted to a thermally stable, pH-insensitive metarhodopsin with lambdamax at 484 nm (M484) and a 43% increase in molar extinction coefficient. Subsequently, short wavelength irradiation of the metarhodopsin photoregenerates some P510; but the absence of an isosbestic point in the cycle of spectral changes is consistent with the presence of smaller amounts of violet- or ultraviolet-sensitive visual pigment(s) that also are converted to a blue-absorb g metarhodopsin. Difference spectra for both P510 and M484 were measured, using hydroxylamine. The 484-nm metarhodopsin is reversibly converted to a form with lambdamax at 363 nm by high concentrations of glycerol. Dark regeneration of rhodopsin in vivo after several minutes exposure of thoroughly dark-adapted animals to full sunlight requires several days.     

Euphausiid visual pigments. The rhodopsins of Euphausia superba and Meganyctiphanes norvegica (Crustacea, Euphausiacea)

The rhabdoms of Euphausia superba contain one digitonin-extractable rhodopsin, lambda max 485 nm. The rhodopsin undergoes unusual pH- dependent spectral changes: above neutrality, the absorbance decreases progressively at 485 nm and rises near 370 nm. This change is reversible and appears to reflect an equilibrium between a protonated and an unprotonated form of the rhodopsin Schiff-base linkage. Near neutral pH and at 10 degrees C, the rhodopsin is partiaLly converted by 420-nm light to a stable 493-nm metarhodopsin. The metarhodopsin is partially photoconverted to rhodopsin by long-wavelength light in the absence of NH2OH; in the presence of NH2OH, it is slowly converted to retinal oxime and opsin. The rhodopsin of Meganyctiphanes norvegica measured in fresh rhabdoms by microspectrophotometry has properties very similar to those of the extracted rhodopsin of E. superba. Its lambda max is 488 nm and it is partially photoconverted by short wavelength irradiation to a stable photoconvertible metarhodopsin similar to that of E. superba. In the presence of light and NH2OH, the M. norvegica metarhodopsin is converted to retinal oxime and opsin. Our results indicate that previous determinations of euphausiid rhodopsin absorbance spectra were incorrect because of accessory pigment contamination.     

Optical activity of octopus metarhodopsins.

The optical activity of octopus rhodopsin, acid metarhodopsin and alkaline metarhodopsin was studied by a sensitive and rapid CD apparatus. For sometime it has been thought that cephalopod metarhodopsins do not have any optical activity associated with their main absorption band. However, the present work shows that acid metarhodopsin in digitonin has a positive CD band at 498 nm and a negative CD band at 436 nm and alkaline metarhodopsin has a negative CD band at 381 nm. Detergent affected the wavelength of the CD peak of the visual pigments though the pattern of the spectrum was similar. From these results it is concluded that the conformation of all-trans retinal in octopus metarhodopsin is influenced by the asymmetric conformation of the protein near the retinal and therefore inducing optical activity.     

Restrictions on rotational and translational diffusion of pigment in the membranes of a rhabdomeric photoreceptor

Individual, isolated rhabdoms from dark-adapted crayfish (Orconectes, Procambarus) were studied with a laterally incident microbeam that could be placed in single stacks of microvilli. Concentration gradients of metarhodopsin along the lengths of microvilli were produced by local bleaches, accomplished by irradiation with small spots of orange light at pH 9 in the presence of glutaraldehyde or formaldehyde. No subsequent redistribution of pigment was observed in the dark, indicating an absence of translational diffusion. On the basis of comparison with other systems, glutaraldehyde, but not formaldehyde (0.75%), would be expected to prevent diffusion of protein in the membrane. Under the same conditions photodichroism is observed, indicating an absence of free Brownian rotation. Photodichroism is larger in glutaraldehyde than in formaldehyde, suggesting that the bifunctional reagent quiets some molecular motion that is present after treatment with formaldehyde. Quantitative comparison of photodichroism with mathematical models indicates that the pigment absorption vectors are aligned within +/- 50 degrees of the microvillar axes and are tilted into the surface of the membrane at an average value of about 20 degrees. The photoconversion of rhodopsin to metarhodopsin is accompanied by an increase in molar extinction of about 20% at the lambda maxand a reorientation of the absorption vector by several degrees. The transition moment either tilts further into the membrane or loses some of its axial orientation, or both. The change in orientation is 3.5 time larger in formaldehyde than in glutaraldehyde.     

The dorsal eye of the dragonfly Sympetrum: specializations for prey detection against the blue sky

Dragonflies of the genus Sympetrum have compound eyes conspicuously divided into dorsal and ventral regions. Using anatomical, optical, electrophysiological, in-vivo photochemical and microspectrophotometrical methods, we have investigated the design and physiology of the dorsal part which is characterized by a pale yellow-orange screening pigment and extremely large facets. The upper part of the yellow dorsal region is a pronounced fovea with interommatidial angles approaching 0.3°, contrasting to the much larger values of 1.5°–2° in the rest of the eye. The dorsal eye part is exclusively sensitive to short wavelengths (below 520 nm). It contains predominantly blue-receptors with a sensitivity maximum at 420 nm, and a smaller amount of UV-receptors. The metarhodopsin of the blue-receptors absorbs maximally at 535 nm. The yellow screening pigment transmits longwavelength light (cut-on 580 nm), which increases the conversion rate from metarhodopsin to rhodopsin (see Fig. 11a). We demonstrate that because of the yellow pigment screen nearly all of the photopigment is in the rhodopsin state under natural conditions, thus maximizing sensitivity. Theoretical considerations show that the extremely long rhabdoms (1.1 mm) in the dorsal fovea are motivated for absorption reasons alone. A surprising consequence of the long rhabdoms is that the sensitivity gain, caused by pumping photopigment into the rhodopsin state, is small. To explain this puzzling fact we present arguments for a mechanism producing a gradient of rhodopsin concentration along the rhabdom, which would minimize saturation of transduction units, and hence improve the signal-to-noise ratio at high intensities. The latter is of special importance for the short integration time and high contrast sensitivity these animals need for spotting small prey at long distances.Abbreviations ERG electroretinogram - R rhodopsin - M metarhodopsin     

Spectral absorption and sensitivity measurements in single ommatidia of the green crab,Carcinus

Summary Rhabdoms of the green crabCarcinus maenas were examined by microspectrophotometry and found to contain a visual pigment with _max at 502–506 nm. Upon irradiation, a stable metarhodopsin formed with unchanged _max and molar extinction coefficient. In the presence of 5% glutaraldehyde the rhabdoms were photobleached. Partial bleaching experiments indicate that in the rhabdoms studied, only one visual pigment was present, with an absorption spectrum appropriate for a hypothetical rhodopsin from Dartnall's (1953) nomogram.Retinular (photoreceptor) cells were studied with microelectrodes. They had negative resting potentials of 30–65 mV and responded to light with depolarizing receptor potentials. All cells had maximum sensitivity at ~493 nm, as did the ERG (electroretinogram). Selective adaptation failed to alter the spectral sensitivity functions of single cells or the ERG. If these spectral sensitivity data are pooled with Wald's (1968), the average sensitivity of the dark-adapted eye is accounted for adequately by the pigment of the rhabdom.The results of this work do not support the hypothesis of Horridge (1967) that each ommatidium ofCarcinus has two color receptors.This work was supported by U.S. P.H.S. grant EY 00222.     

Distribution of rhodopsin and retinochrome in the squid retina

The cephalopod retina contains two kinds of photopigments, rhodopsin and retinochrome. For many years retinochrome has been thought to be localized in the inner segments of the visual cells, whereas rhodopsin is in the outer segments. However, it is now clear that retinochrome can be extracted also from fragments of outer segments. In the dark-adapted retina of Loligo pealei retinochrome is distributed half-and-half in the inner and outer segments. Todarodes pacificus contains much more retinochrome than Loligo, and it is more abundant in the outer than in the inner segments. The outer segments of Loligo contain retinochrome and metarhodopsin in addition to rhodopsin, whether squids are kept in the dark or in the light. But there is extremely little metarhodopsin (about 3% of rhodopsin) even in light-adapted eyes. The inner segments contain only retinochrome, and much less in the light than in the dark. On the other hand, retinochrome in the outer segments increases markedly during light adaptation. These facts suggest the possibility that some retinochrome moves forward from the inner to the outer segments during light adaptation and there reacts with metarhodopsin to promote regeneration of rhodopsin.     

Visual pigment,dark adaptation and rhodopsin renewal in the eye of Pontoporeia affinis (Crustacea,Amphipoda)

The benthic amphipod Pontoporeia affinis lives in the Baltic sea and in northern European lakes in an environment where very little light is available for vision. The eyes, consisting of 40–50 ommatidia, are correspondingly modified. Microspectrophotometric recordings on isolated eyes show the presence of at least two kinds of screening pigments in the ommatidia with maxima at 540–580 nm and 460–500 nm. Difference spectra obtained from the rhabdoms after exposure to red and blue light, respectively, give evidence of a single rhodopsin with its maximum at 548 nm and a 500-nm metarhodopsin. In ERG recordings sensitivity in the dark-adapted state, after saturating exposures to blue and to red light, stabilizes at levels determined by the rhodopsin concentration. No change is observed during 10–14 h after the beginning of dark adaptation. However, using animals pre-exposed with a strong red light and then kept in darkness, it is found that after a delay of 20–40 h sensitivity of the dark-adapted eye begins to increase and finally, after 5–6 days reaches a level corresponding to 100% rhodopsin. Thus, a slow renewal of rhodopsin appears to occur in darkness, where a photoisomerization of metarhodopsin is excluded.Abbreviations ERG electroretinogram - IR infrared - MSP microspectrophotometry     

Light-induced conformational changes of rhodopsin probed by fluorescent alexa594 immobilized on the cytoplasmic surface

A novel fluorescence method has been developed for detecting the light-induced conformational changes of rhodopsin and for monitoring the interaction between photolyzed rhodopsin and G-protein or arrestin. Rhodopsin in native membranes was selectively modified with fluorescent Alexa594-maleimide at the Cys(316) position, with a large excess of the reagent Cys(140) that was also derivatized. Modification with Alexa594 allowed the monitoring of fluorescence changes at a red excitation light wavelength of 605 nm, thus avoiding significant rhodopsin bleaching. Upon absorption of a photon by rhodopsin, the fluorescence intensity increased as much as 20% at acidic pH with an apparent pK(a) of approximately 6.8 at 4 degrees C, and was sensitive to the presence of hydroxylamine. These findings indicated that the increase in fluorescence is specific for metarhodopsin II. In the presence of transducin, a significant increase in fluorescence was observed. This increase of fluorescence emission intensity was reduced by addition of GTP, in agreement with the fact that transducin enhances the formation of metarhodopsin II. Under conditions that favored the formation of a metarhodopsin II-Alexa594 complex, transducin slightly decreased the fluorescence. In the presence of arrestin, under conditions that favored the formation of metarhodopsin I or II, a phosphorylated, photolyzed rhodopsin-Alexa594 complex only slightly decreased the fluorescence intensity, suggesting that the cytoplasmic surface structure of metarhodopsin II is different in the complex with arrestin and transducin. These results demonstrate the application of Alexa594-modified rhodopsin (Alexa594-rhodopsin) to continuously monitor the conformational changes in rhodopsin during light-induced transformations and its interactions with other proteins.     

Photopigment and receptor properties in Drosophila compound eye and ocellar receptors

A review of the spectral sensitivity and the rhodopsin and metarhodopsin characteristics in three compound eye receptor types (R1-6, R7, and R8) and ocellar receptors is presented (Fig. 1). Photopigment properties were determined from measures of conversion efficiency. The photopigments of R1-6 were studied using in vivo microspectrophotometry in the deep pseudopupil of white-eyed flies. These studies yielded a refined estimate of the R1-6 metarhodopsin spectrum (Fig. 2). The quantum efficiency relative to the spectral sensitivity estimate of the rhodopsin spectrum was factored out. The quantum efficiency of rhodopsin is about 1.75 times that of metarhodopsin. The peak absorbance of metarhodopsin was estimated to be about 2.6 times that of rhodopsin. The mechanism of the two-peaked R1-6 spectral sensitivity and metarhodopsin spectrum is discussed in terms of evidence that there is only one rhodopsin in R1-6 and that vitamin A deprivation preferentially lowers ultraviolet sensitivity. The prolonged depolarizing afterpotential is reviewed from the standpoint of the internal transmitter hypothesis of visual excitation. A careful comparison of the intensity-responsivity for photopigment conversion and its adaptional consequences is made (Fig. 3).     

Microspectrophotometry of single rhabdoms in the retina of the honeybee drone (Apis mellifera male)

The relative absorption spectra of the bistable photopigment of single rhabdoms from the dorsal region of the retina of the honeybee drone were obtained using slices of retina fixed in glutaraldehyde; less accurate measurements on unfixed tissue gave difference spectra that were similar to those for fixed retinae. The method used was based on measurements of absorbance changes during saturating adaptations of the visual pigment to different monochromatic lights. It is similar to previous methods based on measurements of difference spectra amplitudes, but is simpler to use and more accurate. The predominant pigment has states that absorb maximally at 446 (rhodopsin) and 505 nm (metarhodopsin). In addition, there is a small amount of another pigment whose two states absorb maximally at approximately 340 (UV) and 460 nm.     

Heat shock protein 70 and heat shock protein 90 expression in light- and dark-adapted adult octopus retinas

Light- and dark-adaptation leads to changes in rhabdom morphology and photopigment distribution in the octopus retina. Molecular chaperones, including heat shock proteins (Hsps), may be involved in specific signaling pathways that cause changes in photoreceptor actin- and tubulin-based cytoskeletons and movement of the photopigments, rhodopsin and retinochrome. In this study, we used immunoblotting, in situ RT-PCR, immunofluorescence and confocal microscopy to localize the inducible form of Hsp70 and the larger Hsp90 in light- and dark-adapted and dorsal and ventral halves of adult octopus retinas. The Hsps showed differences in distribution between the light and dark and in dorsal vs. ventral position in the retina. Double labeling confocal microscopy co-localized Hsp70 with actin and tubulin, and Hsp90 with the photopigment, retinochrome. Our results demonstrate the presence of Hsp70 and Hsp90 in otherwise non-stressed light- and dark-adapted octopus retinas. These Hsps may help stabilize the cytoskeleton, important for rhabdom structure, and are perhaps involved in the redistribution of retinochrome in conditions of light and dark.     

Photopigment and receptor properties in Drosophila compound eye and ocellar receptors

A review of the spectral sensitivity and the rhodopsin and metarhodopsin characteristics in three compound eye receptor types (R1–6, R7, and R8) and ocellar receptors is presented (Fig. 1). Photopigment properties were determined from measures of conversion efficiency. The photopigments of R1–6 were studied using in vivo microspectrophotometry in the deep pseudopupil of white-eyed flies. These studies yielded a refined estimate of the R1–6 metarhodopsin spectrum (Fig. 2). The quantum efficiency relative to the spectral sensitivity estimate of the rhodopsin spectrum was factored out. The quantum efficiency of rhodopsin is about 1.75 times that of metarhodopsin. The peak absorbance of metarhodopsin was estimated to be about 2.6 times that of rhodopsin. The mechanism of the two-peaked R1–6 spectral sensitivity and metarhodopsin spectrum is discussed in terms of evidence that there is only one rhodopsin in R1–6 and that vitamin A deprivation preferentially lowers ultraviolet sensitivity. The prolonged depolarizing afterpotential is reviewed from the standpoint of the internal transmitter hypothesis of visual excitation. A careful comparison of the intensity-responsivity for photopigment conversion and its adaptional consequences is made (Fig. 3).     

The visual pigment of a stomatopod crustacean,Squilla empusa

Summary Stomatopod crustaceans are visually active animals which have large, mobile compound eyes of unique design. Aspects of their ecology and behavior suggest they may be able to discriminate hues. Isolated rhabdoms of the squillid stomatopod,Squilla empusa, were investigated using microspectrophotometry and fluorometry. A single rhodopsin, of _max507 nm, exists in the main rhabdom. Its stable metarhodopsin, with _max503 nm, possesses typical arthropod fluorescence characteristics. No evidence was found for a visual pigment with peak absorption in the ultraviolet. Vision in this animal might therefore be monochromatic.Abbreviation ASW artificial sea water     

Constraints on the conformation of the cytoplasmic face of dark-adapted and light-excited rhodopsin inferred from antirhodopsin antibody imprints

Rhodopsin is the best-understood member of the large G protein-coupled receptor (GPCR) superfamily. The G-protein amplification cascade is triggered by poorly understood light-induced conformational changes in rhodopsin that are homologous to changes caused by agonists in other GPCRs. We have applied the "antibody imprint" method to light-activated rhodopsin in native membranes by using nine monoclonal antibodies (mAbs) against aqueous faces of rhodopsin. Epitopes recognized by these mAbs were found by selection from random peptide libraries displayed on phage. A new computer algorithm, FINDMAP, was used to map the epitopes to discontinuous segments of rhodopsin that are distant in the primary sequence but are in close spatial proximity in the structure. The proximity of a segment of the N-terminal and the loop between helices VI and VIII found by FINDMAP is consistent with the X-ray structure of the dark-adapted rhodopsin. Epitopes to the cytoplasmic face segregated into two classes with different predicted spatial proximities of protein segments that correlate with different preferences of the antibodies for stabilizing the metarhodopsin I or metarhodopsin II conformations of light-excited rhodopsin. Epitopes of antibodies that stabilize metarhodopsin II indicate conformational changes from dark-adapted rhodopsin, including rearrangements of the C-terminal tail and altered exposure of the cytoplasmic end of helix VI, a portion of the C-3 loop, and helix VIII. As additional antibodies are subjected to antibody imprinting, this approach should provide increasingly detailed information on the conformation of light-excited rhodopsin and be applicable to structural studies of other challenging protein targets.     

Further characterization of the lipid-depleted bovine rhodopsin obtained by cholate-ammonium sulfate fractionation

The rhodopsin preparation obtained by the method of ammonium sulfate fractionation contained 3–6 mol phospholipid and about 18 mol cholate per mol rhodopsin. The purified rhodopsin had 74% helical structure and showed a visible CD spectrum different from that of rhodopsin in the membrane. The rhodopsin was stable below but denatured gradually above 20°C. The lifetime of metarhodopsin I was long in this preparation. Regeneration capacity was low and only 30% of the original rhodopsin was regenerable by addition of 11-cis-retinal after bleaching.50 mol of phosphatidylcholine were maximally bound to 1 mol rhodopsin when the purified rhodopsin was mixed with phosphatidylcholine in 0.5% cholate. The rhodopsin recombined with lipid had properties similar to those of the original rhodopsin in the membrane. Exchange of cholate for other detergents was easily performed by dialysis. The rhodopsin preparation in which cholate was exchanged for digitonin gave almost the same CD, thermal stability and regenerability as those of a native rhodopsin in the membrane but metarhodopsin I still retained its long lifetime.