Molecular mechanisms of receptor-potential generation by the photoreceptor. III. Conformational transition responsible for the tail end of the photoresponse of an artificial lipid membrane modified by fragments of the external segments of rods

The kinetics of photoinduced changes of protein fluorescence of cattle visual pigment was studied in the presence of hydroxylamine. The rate constant of fluorescence increase is proportional to NH2OH concentration when it is less than 0.4 M. It reaches the maximal magnitude (3.3 +/- 1 sec-1) at higher hydroxylamine concentration. Fluorescence increase rate is controlled by the rate of chemical reaction of rhodopsin with hydroxylamine. It is limited by conformational rearrangement of opsin. This rearrangement does not induce absorbance spectrum change of visual pigment, but confers to it the capability to react with NH2OH and NaBH4. Kinetic parameters of this rearrangement (tau 20 degrees C approximately 300 msec, Eact = 19 +/- 2 kcal/mole) coincide with kinetic parameters of diminishing of the photoresponse of artificial lipid membrane modified by fragments of rod outer segments in the temperature range studied (+2 divided by +25 degrees C).     

Kinetics of permeability changes induced by electric impulses in chromaffin granules

Summary Electric field pulses, ranging in intensity from 20 to 50 kV/cm and in duration from 10 to 40 sec, caused a transient increase in the membrane permeability of chromaffin granules from the bovine adrenal medulla, that led to partial release of granule soluble constituents. This transient permeability change was long-lived, as compared to the pulse duration, and the main part of material efflux occurred after the termination of the pulse. During the latter phase the temporarily increased permeability decayed to its original value, in the absence of the electric field. This indicated that the structural perturbation induced in the membrane was transient and apparently reversible. The release event was characterized by a field-dependent permeability coefficient ranging from 2×10^–4 cm/sec at 30 kV/cm to 3×10^–3 cm/sec at 50 kV/cm. The resealing process of the membrane could be described by two relaxation times, both of which decreased with increasing field strength. ₁ varied from about 3.0 msec at 30 kV/cm to less than 2.0 msec at 50 kV/cm, while ₂ varied from about 100 to about 40 msec in the same interval of field strength. The distribution in the degree of filling of granules that had been partially depleted by an electric field pulse indicated that the population could be considered homogeneous with respect to release.     

Fluorescence signals in ANS-stained squid giant axons during voltage-clamp

Summary An analysis is presented of the changes in fluorescence intensity, associated with nerve stimulation, of 1-anilinonaphthalene-8-sulfonate (ANS) injected in squid axons. A preliminary and qualitative account of the physiological modifications produced by the ANS injection is also given. The time course of the fluorescence intensity during the first 300 sec following the onset of voltage-clamp is shown to be exponential with a time constant of about 35 msec, fairly independent of the amplitude and sign of the applied voltage, the intensity increasing during hyperpolarizations and decreasing during depolarizations. Data are presented on the relationship between the amplitude of the changes in fluorescence intensity and the voltage applied, the amplitude of the changes associated with depolarizations being measured at the time of occurence of the peak inward current. The interpretation of the changes in fluorescence intensity in terms of electrophoretic effects or as being due to a direct effect of the electric field upon the quantum yield of ANS fluorescence, is hardly compatible with the results of our present analysis.     

Formation,structure, and spectrophotometry of air-water interface films containing rhodopsin

Summary Air-water interface films of cattle rhodopsin and defined lipids are formed without the use of organic solvents by a method in which vesicle membranes consisting of egg phosphatidyl choline and purified rhodopsin are osmotically shocked at the interface. Lipid and protein molecules organize as insoluble films at the interface. The structure of these films varies with the lipid to protein mole ratio of the source vesicle membranes. Electron microscopic observations reveal that films formed with membranes of 1501 mole ratio consist of nonoverlapping, randomly distributed vesicle membrane fragments separated by a lipid monolayer. These membrane fragments exist as single sheets on the water surface and occupy approximately 35% of this surface. Essentially all the rhodopsin molecules at the interface are spectroscopically intact and are contained within the membrane fragments. The visible absorption spectrum of the interface films is identical to that of suspensions of rod disc membranes. Moreover, flash illumination of rhodopsin in air-dried multilayers formed from the interface films results in the formation of a stable MetarhodopsinI intermediate (_max480 nm) which can be fully bleached by increasing the relative humidity of the multilayers or can be photoconverted into rhodopsin and, presumably, isorhodopsin. Furthermore, rhodopsin is chemically regenerable at the air-water interface. Bleached rhodopsin can generate dark rhodopsin at the interface in the presence of 11-cis retinal in the aqueous subphase. Thus, the spectroscopic structure and the chemical regenerability function of rhodopsin in these interface films are indistinguishable from those exhibited by the protein in intact rod disc membranes.     

Mechanism of interaction of the cyanine dye DiS−C3-(5) with renal brush-border vesicles

Summary The equilibrium binding mechanism and kinetics of binding of diS–C₃-(5) (3,3-dipropylthiodicarbocyanine iodide) to rabbit renal brush-border membrane vesicles (BBMV) were examined using steady-state and time-resolved fluorescence, and fluorescence stopped-flow methods. In aqueous solution, diS–C₃-(5) exists as a monomer at concentrations <5 m with fluorescence emission peak at 670 nm (excitation 622 nm), anisotropyr=0.102, and lifetime =1.2 nsec (23°C). Upon addition of increasing BBMV (voltage clamped to 0 mV using K⁺/valinomycin), the 670 nm emission peak decreases, corresponding to formation of a nonfluorescent membrane dimer, and subsequently a new emission peak at 695 nm increases, corresponding to membrane monomer. Dynamic depolarization studies show that aqueous diS–C₃-(5) rotation is unhindered with a rotational rateR=0.57 nsec^–1 while membrane monomer is hindered with steady-state anisotropyr=0.190, lifetime =2.1 nsec,R=0.58 nsec^–1 and limiting anisotropyr =0.11. Based on equilibrium fluorescence titrations, the membrane monomer-dimer (M-D) dissociation constant,K _d=[M]²/[D][BBMV], is 0.0013, where BBMV is expressed as membrane phospholipid concentration. Three distinct kinetic processes are identified by stopped-flow experiments in which BBMV are mixed with diS–C₃-(5). There is rapid binding of diS–C₃-(5) to the membrane to form bound monomer with a 6-msec exponential time constant. The membrane monomer at the membrane outer surface then aggregates to form bound dimer at the outer surface with a concentration independent time constant of 30 msec. The overall dimerization reaction probably consists of a rate-limiting reorientation process (30 msec) followed by a rapid dimerization which occurs on a nanosecond time scale. Finally, there is a 0.8 to 1 sec translocation of membrane dimer between symmetric sites at the inner and outer membrane surfaces. The translocation reaction is the step which is probably sensitive to changes in transmembrane electrical potential.     

Molecular mechanisms of receptor. II. Identification of the conformational transition of rhodopsin responsible for the leading edge of the photoresponse of artificial lipid membranes modified by fragments of the outer segment of rods

Kinetic parameters of photoinduced permeability increase of artificial lipid membranes, modified by ROS fragments (tau20 degrees C = 20 mesec Ea = 33 +/- 2 kcal/mole) coincides with appropriate parameters of photoinduced protein fluorescence intensity decrease and ROS fragments absorption spectra change (metarhodopsin I leads leads to metarhodopsin II transition). Hydroxylamine accelerates this process, its rate is proportional to hydroxylamine at concentrations lower than 0.6 M.     

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.     

Molecular mechanisms of receptor-potential generation by the photoreceptor. I. Photic induction of a change in the permeability of artificial lipid membranes modified by photoreceptor membrane fragments

Artificial lipid membranes modified by ultrasonic fragments of rod outer segments increase their conduction in response to illumination. Conduction increase is followed by the spontaneous foll in the dark to the unitial or somewhat higher level. The time constant of conduction increase was about 30 msec at room temperature, the constant of its following drop was about 300 msec; the activation energy of the last process was 19 +/- 3 kcal/mole.     

Binding of rhodopsin and rhodopsin analogues to transducin,rhodopsin kinase and arrestin-1

AIM: To investigate the interaction of reconstituted rhodopsin, 9-cis-retinal-rhodopsin and 13-cis-retinal-rhodopsin with transducin, rhodopsin kinase and arrestin-1. METHODS: Rod outer segments(ROS) were isolated from bovine retinas. Following bleaching of ROS membranes with hydroxylamine, rhodopsin and rhodopsin analogues were generated with the different retinal isomers and the concentration of the reconstituted pigments was calculated from their UV/visible absorption spectra. Transducin and arrestin-1 were purified to homogeneity by column chromatography, and an enriched-fraction of rhodopsin kinase was obtainedby extracting freshly prepared ROS in the dark. The guanine nucleotide binding activity of transducin was determined by Millipore filtration using β,γ-imido-(3H)-guanosine 5'-triphosphate. Recognition of the reconstituted pigments by rhodopsin kinase was determined by autoradiography following incubation of ROS membranes containing the various regenerated pigments with partially purified rhodopsin kinase in the presence of(γ-32P) ATP. Binding of arrestin-1 to the various pigments in ROS membranes was determined by a sedimentation assay analyzed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis. RESULTS: Reconstituted rhodopsin and rhodopsin analogues containing 9-cis-retinal and 13-cis-retinal rendered an absorption spectrum showing a maximum peak at 498 nm, 486 nm and about 467 nm, respectively, in the dark; which was shifted to 380 nm, 404 nm and about 425 nm, respectively, after illumination. The percentage of reconstitution of rhodopsin and the rhodopsin analogues containing 9-cis-retinal and 13-cis-retinal was estimated to be 88%, 81% and 24%, respectively. Although only residual activation of transducin was observed in the dark when reconstituted rhodopsin and 9-cis-retinal-rhodopsin was used, the rhodopsin analogue containing the 13-cis isomer of retinal was capable of activating transducin independently of light. Moreover, only a basal amount of the reconstituted rhodopsin and 9-cis-retinal-rhodopsin was phosphorylated by rhodopsin kinase in the dark, whereas the pigment containing the 13-cis-retinal was highly phosphorylated by rhodopsin kinase even in the dark. In addition, arrestin-1 was incubated with rhodopsin, 9-cis-retinal-rhodopsin or 13-cis-retinal-rhodopsin. Experiments were performed using both phosphorylated and non-phosphorylated regenerated pigments. Basal amounts of arrestin-1 interacted with rhodopsin, 9-cis-retinal-rhodopsin and 13-cis-retinal-rhodopsin under dark and light conditions. Residual arrestin-1 was also recognized by the phosphorylated rhodopsin and phosphorylated 9-cis-retinal-rhodopsin in the dark. However, arrestin-1 was recognized by phosphorylated 13-cis-retinal-rhodopsin in the dark. As expected, all reformed pigments were capable of activating transducin and being phosphorylated by rhodopsin kinase in a lightdependent manner. Additionally, all reconstituted photolyzed and phosphorylated pigments were capable of interacting with arrestin-1. CONCLUSION: In the dark, the rhodopsin analogue containing the 13-cis isomer of retinal appears to fold in a pseudo-active conformation that mimics the active photointermediate of rhodopsin.     

The bilayer enhances rhodopsin kinetic stability in bovine rod outer segment disk membranes

Rhodopsin is a kinetically stable protein constituting >90% of rod outer segment disk membrane protein. To investigate the bilayer contribution to rhodopsin kinetic stability, disk membranes were systematically disrupted by octyl-β-D-glucopyranoside. Rhodopsin kinetic stability was examined under subsolubilizing (rhodopsin in a bilayer environment perturbed by octyl-β-D-glucopyranoside) and under fully solubilizing conditions (rhodopsin in a micelle with cosolubilized phospholipids). As determined by DSC, rhodopsin exhibited a scan-rate-dependent irreversible endothermic transition at all stages of solubilization. The transition temperature (T_m) decreased in the subsolubilizing stage. However, once the rhodopsin was in a micelle environment there was little change of the T_m as the phospholipid/rhodopsin ratio in the mixed micelles decreased during the fully solubilized stage. Rhodopsin thermal denaturation is consistent with the two-state irreversible model at all stages of solubilization. The activation energy of denaturation (E_act) was calculated from the scan rate dependence of the T_m and from the rate of rhodopsin thermal bleaching at all stages of solubilization. The E_act as determined by both techniques decreased in the subsolubilizing stage, but remained constant once fully solubilized. These results indicate the bilayer structure increases the E_act to rhodopsin denaturation.     

Interaction of bovine rhodopsin with calcium ions

The formation of metarhodopsin II in various bovine rhodopsin preparations (rod outer segment (ROS) suspensions and rhodopsin-detergent solutions) was measured by means of flash spectrophotometry. The half-lifetime and formation of metarhodopsin II in ROS did not depend on the calcium concentration in the range of less than 10^–9 M (using EGTA or EDTA) to 15×10^–3 M calcium at pH values of 5.0, 7.1, and 9.0 (Table 1).The regeneration of rhodopsin from opsin by adding 11-cis retinal to ROS-suspensions and rhodopsin digitonin solutions was measured spectrophotometrically. It was not substantially different in either saline, one containing less than 10^–7 M calcium (by adding EGTA), the other containing 10^–3 M calcium (Table 2).Abbreviations A absorption - A absorption change - CTAB N-Cetyl-N,N,N-trimethylammoniumbromide - E₇₀₀ extinction at =700 nm - EDTA ethylenediamine-NNNN-tetraacetic acid - EGTA 2,2-ethylenedioxybis [ethyliminodi (acetic acid)] - MI metarhodopsin I - MII metarhodopsin II - Rh rhodopsin - ROS rod outer segment This work is based upon a Ph. D. dissertation (Nöll, 1974) and was presented in part at the Jahrestagung der Deutschen Gesellschaft für Biophysik, Freiburg, Germany, October 1974     

The molecular weight of rhodopsin and the nature of the rhodopsin-digitonin complex

The sedimentation behavior of aqueous solutions of digitonin and of cattle rhodopsin in digitonin has been examined in the ultracentrifuge. In confirmation of earlier work, digitonin was found to sediment as a micelle (D-1) with an s₂₀ of about 6.35 Svedberg units, and containing at least 60 molecules. The rhodopsin solutions sediment as a stoichiometric complex of rhodopsin with digitonin (RD-1) with an s₂₀ of about 9.77 Svedberg units. The s₂₀ of the RD-1 micelle is constant between pH 6.3 and 9.6, and in the presence of excess digitonin. RD-1 travels as a single boundary also in the electrophoresis apparatus at pH 8.5, and on filter paper at pH 8.0. The molecular weight of the RD-1 micelle lies between 260,000 and 290,000. Of this, only about 40,000 gm. are due to rhodopsin; the rest is digitonin (180 to 200 moles). Comparison of the relative concentrations of RD-1 and retinene in solutions of rhodopsin-digitonin shows that RD-1 contains only one retinene equivalent. It can therefore contain only one molecule of rhodopsin with a molecular weight of about 40,000. Cattle rhodopsin therefore contains only one chromophore consisting of a single molecule of retinene. It is likely that frog rhodopsin has a similar molecular weight and also contains only one chromophore per molecule. The molar extinction coefficient of rhodopsin is therefore identical with the extinction coefficient per mole of retinene (40,600 cm.² per mole) and the E(1 per cent, 1 cm., 500 mµ) has a value of about 10. Rhodopsin constitutes about 14 per cent of the dry weight, and 3.7 per cent of the wet weight of cattle outer limbs. This corresponds to about 4.2 x 10⁶ molecules of rhodopsin per outer limb. The rhodopsin content of frog outer limbs is considerably higher: about 35 per cent of the dry weight, and 10 per cent of the wet weight, corresponding to about 2.1 x 10⁹ molecules per outer limb. Thus the frog outer limb contains about five hundred times as much rhodopsin as the cattle outer limb. But the relative volumes of these structures are such that the ratio of concentrations is only about 2.5 to 1 on a weight basis. Rhodopsin accounts for at least one-fifth of the total protein of the cattle outer limb; for the frog, this value must be higher. The extinction (K₅₀₀) along its axis is about 0.037 cm.² for the cattle outer limb, and about 0.50 cm.² for the frog outer limb.     

Calcium regulates the rate of rhodopsin disactivation and the primary amplification step in visual transduction

The kinetics of the light-induced activation of transducin as well as the subsequent disactivation process can be monitored by means of a specific light scattering transient PA. In this communication it is demonstrated that the rate of transducin disactivation is calcium dependent, increasing when the calcium concentration is decreased. As a consequence of the accelerated recovery in low calcium, the time to the peak of the transducin activation process is shortened and the gain of the primary amplification step, i.e. the number of transducin molecules activated per bleached rhodopsin, is reduced. Experiments using hydroxylamine as an artificial quencher of rhodopsin activity suggest that calcium acts upon rhodopsin kinase and not upon the rate of the GTPase. This would indicate that calcium may control visual adaptation not only by regulating guanine cyclase activity, but also by affecting the primary step in the transduction cascade, the rhodopsin-transducin coupling.     

The action of enzymes on rhodopsin

The effects have been examined of chymotrypsin, pepsin, trypsin, and pancreatic lipase on cattle rhodopsin in digitonin solution. The digestion of rhodopsin by chymotrypsin was measured by the hydrolysis of peptide bonds (formol titration), changes in pH, and bleaching. The digestion proceeds in two stages: an initial rapid hydrolysis which exposes about 30 amino groups per molecule, without bleaching; superimposed on a slower hydrolysis which exposes about 50 additional amino groups, with proportionate bleaching. The chymotryptic action begins at pH about 6.0 and increases logarithmically in rate to pH 9.2. Trypsin and pepsin also bleach rhodopsin in solution. A preparation of pancreatic lipase bleached it slightly, but no more than could be explained by contamination with proteases. In digitonin solution each rhodopsin molecule is associated in a micelle with about 200 molecules of digitonin; yet the latter do not appear to hinder enzyme action. It is suggested that the digitonin sheath is sufficiently fluid to be penetrated on collision with an enzyme molecule; and that once together the enzyme and substrate are held together by intermolecular attractive forces, and by the "cage effect" of bombardment by surrounding solvent molecules. The two stages of chymotryptic digestion of rhodopsin may correspond to an initial rapid fragmentation, such as has been observed with many proteinases and substrates; superimposed upon a slower digestion of the fragments. Since the first phase involves no bleaching, this may mean that rhodopsin can be broken into considerably smaller fragments without loss of optical properties.     

The rhodopsin system of the squid

Squid rhodopsin (λ_max 493 mµ)—like vertebrate rhodopsins—contains a retinene chromophore linked to a protein, opsin. Light transforms rhodopsin to lumi- and metarhodopsin. However, whereas vertebrate metarhodopsin at physiological temperatures decomposes into retinene and opsin, squid metarhodopsin is stable. Light also converts squid metarhodopsin to rhodopsin. Rhodopsin is therefore regenerated from metarhodopsin in the light. Irradiation of rhodopsin or metarhodopsin produces a steady state by promoting the reactions, See PDF for Equation Squid rhodopsin contains neo-b (11-cis) retinene; metarhodopsin all-trans retinene. The interconversion of rhodopsin and metarhodopsin involves only the stereoisomerization of their chromophores. Squid metarhodopsin is a pH indicator, red (λ_max 500 mµ) near neutrality, yellow (λ_max 380 mµ) in alkaline solution. The two forms—acid and alkaline metarhodopsin—are interconverted according to the equation, Alkaline metarhodopsin + H⁺ acid metarhodopsin, with pK 7.7. In both forms, retinene is attached to opsin at the same site as in rhodopsin. However, metarhodopsin decomposes more readily than rhodopsin into retinene and opsin. The opsins apparently fit the shape of the neo-b chromophore. When light isomerizes the chromophore to the all-trans configuration, squid opsin accepts the all-trans chromophore, while vertebrate opsins do not and hence release all-trans retinene. Light triggers vision by affecting directly the shape of the retinene chromophore. This changes its relationship with opsin, so initiating a train of chemical reactions.     

Photoenergetics of octopus rhodopsin

The enthalpy changes associated with each of the major steps in the photoconversion of octopus rhodopsin have been measured by direct photocalorimetry. Formation of the primary photoproduct (bathorhodopsin) involves energy uptake of about 130 kJ/mol, corresponding to storage of over 50% of the exciting photon energy, and is comparable to the energy storage previously observed in bovine rhodopsin. Subsequent intermediates involve the step-wise dissipation of this energy to give the physiological end-product (acid metarhodopsin) at a level only slightly above the parent rhodopsin. No significant differences in energetics are observed between rhodopsin in microvilli membrane suspensions or detergent dispersions. Use of different buffer systems in the calorimetric experiments shows that conversion of rhodopsin to acid metarhodopsin involves no light-induced protonation change, whereas alkali metarhodopsin photoproduction occurs with the release of one proton per molecule and an additional enthalpy increase of about 50 kJ/mol. Van't Hoff analysis of the effect of temperature on the reversible metarhodopsin equilibrium gives an enthalpy for the acid alkali transition consistent with this calorimetric result, and the proton release is confirmed by direct observation of light-induced pH changes. Acid-base titration of metarhodopsin yields an apparent pK of 9.5 for this transition, though the pH profile deviates slightly from ideal titration behaviour. We suggest that a high energy primary photoproduct is an obligatory feature of efficient biological photodetectors, as opposed to photon energy transducers, and that the similarity at this stage between cephalopod and vertebrate rhodopsins represents either convergent evolution at the molecular level or strong conservation of a crucial functional characteristic.     

Principal absorption axes of rhodopsin and prelumirhodopsin

The experimental data on the absorption of plane polarized light by a solution of cattle rhodopsin at –196 C have been theoretically analysed to model the directional absorption properties of rhodopsin and prelumirhodopsin. It has been found that rhodopsin and prelumirhodopsin are planar absorbers having ratios of about 1007 and 1004, respectively, between the extinction coefficients along the long axis and perpendicular to it. These results support that the chromophore in prelumirhodopsin is more linear than the chromophore in rhodopsin.Work partially supported by Department of Science and Technology (India)Associated with the Biochemistry Cell     

LEDGF(1-326) decreases P23H and wild type rhodopsin aggregates and P23H rhodopsin mediated cell damage in human retinal pigment epithelial cells

Background

P23H rhodopsin, a mutant rhodopsin, is known to aggregate and cause retinal degeneration. However, its effects on retinal pigment epithelial (RPE) cells are unknown. The purpose of this study was to determine the effect of P23H rhodopsin in RPE cells and further assess whether LEDGF_1-326, a protein devoid of heat shock elements of LEDGF, a cell survival factor, reduces P23H rhodopsin aggregates and any associated cellular damage.

Methods

ARPE-19 cells were transiently transfected/cotransfected with pLEDGF_1-326 and/or pWT-Rho (wild type)/pP23H-Rho. Rhodopsin mediated cellular damage and rescue by LEDGF_1-326 was assessed using cell viability, cell proliferation, and confocal microscopy assays. Rhodopsin monomers, oligomers, and their reduction in the presence of LEDGF_1-326 were quantified by western blot analysis. P23H rhodopsin mRNA levels in the presence and absence of LEDGF_1-326 was determined by real time quantitative PCR.

Principal Findings

P23H rhodopsin reduced RPE cell viability and cell proliferation in a dose dependent manner, and disrupted the nuclear material. LEDGF_1-326 did not alter P23H rhodopsin mRNA levels, reduced its oligomers, and significantly increased RPE cell viability as well as proliferation, while reducing nuclear damage. WT rhodopsin formed oligomers, although to a smaller extent than P23H rhodopsin. Further, LEDGF_1-326 decreased WT rhodopsin aggregates.

Conclusions

P23H rhodopsin as well as WT rhodopsin form aggregates in RPE cells and LEDGF_1-326 decreases these aggregates. Further, LEDGF_1-326 reduces the RPE cell damage caused by P23H rhodopsin. LEDGF_1-326 might be useful in treating cellular damage associated with protein aggregation diseases such as retinitis pigmentosa.     

Flash photolysis of rhodopsin in rabbit

Flash photolysis of rhodopsin in rabbit's retina has been analysed theoretically, and the results are found to be in good agreement with the experimental results of Hagins (1957). We have also obtained the variation of relative concentrations of rhodopsin, lumirhodopsin, isorhodopsin and metarhodopsin I during the period of the flash corresponding to two different intensities of the flash. It has been found that the quantum efficiencies of conversion of lumirhodopsin into rhodopsin and isorhodopsin will lie in the range 0.24–0.45 and 0.20–0.44 respectively; quantum efficiencies of conversion of metarhodopsin I into rhodopsin and isorhodopsin are found to have values greater than 0.52 and 0.45 respectively and the quantum efficiency of conversion of isorhodopsin into lumirhodopsin has been found to be approximately 0.865. Also the maximum value of the rate constant of the reaction metarhodopsin Imetarhodopsin II at 37 C has been determined in decerebrated eye and it has been found that it is of the same order as found by Pugh (1975) in the case of human eye.Work partially supported by Department of Science and Technology