Metarhodopsin II Research Articles

A role for phospholipid polyunsaturation in modulating membrane protein function.

Visual transduction is one of the best characterized G protein--coupled signalling systems. In addition, about 50% of the disk membrane phospholipid acyl chains are 22:6n-3, making this system ideal for determining the role of polyunsaturation in modulating membrane-signalling systems. The extent of formation of metarhodopsin II (MII), the G protein--activating photointermediate of rhodopsin, was studied in phospholipid vesicles composed of a variety of phosphatidylcholines, differing in their acyl chain composition at the sn-2 position. The amount of MII formed increased progressively with the level of acyl chain unsaturation at the sn-2 position. The effect of added cholesterol was to reduce the amount of MII formed. The acyl chain packing free volume of the rhodopsin containing lipid vesicles was characterized by a fractional volume parameter fv derived from measurements of the time-resolved fluorescence anisotropy decay of the hydrophobic membrane probe 1,6-diphenyl-1,3,5-hexatriene. The relationship among sn-2 acyl chain unsaturation, cholesterol content, and MII formation is explained on the basis of variation in fv with bilayer lipid composition and a novel model for the packing of phospholipids containing polyenoic acyl chains, such as 22:6n-3.

Identification of glutamic acid 113 as the Schiff base proton acceptor in the metarhodopsin II photointermediate of rhodopsin.

In order to investigate the molecular mechanism of rhodopsin photoactivation, site-directed mutants of bovine rhodopsin were studied by Fourier-transform infrared (FTIR) difference spectroscopy. Rhodopsin mutants E113D and E113A were prepared in which the retinylidene Schiff base counterion, Glu113, was replaced by Asp and Ala, respectively. FTIR difference spectra were recorded and compared with spectra of recombinant native rhodopsin. Both mutant pigments formed photoproducts at 0 degrees C with vibrational absorption bands typical of the metarhodopsin II (MII) state of rhodopsin. The FTIR difference spectrum of E113D was nearly identical to that of rhodopsin. A positive band at 1712 cm-1 caused by the protonation of an internal carboxylic acid in rhodopsin was shifted slightly to 1709 cm-1 in mutant E113D. E113A was studied at acidic pH in the presence of chloride as an inorganic counterion to the protonated Schiff base. The 1712-cm-1 (1709-cm-1) band was absent in the FTIR difference spectrum of mutant E113A. Therefore, we have assigned the 1712-cm-1 absorbance band to the C = O stretching vibration of protonated Glu113 in MII of rhodopsin. These results show that the Schiff base counterion of rhodopsin, the carboxylate side chain of Glu113, becomes protonated during MII formation.

A conserved carboxylic acid group mediates light-dependent proton uptake and signaling by rhodopsin.

A carboxylic acid residue is conserved at the cytoplasmic border of the third transmembrane segment among nearly all G protein-coupled receptors. In the visual receptor rhodopsin, replacement of the conserved Glu134 by a neutral glutamine results in enhanced transducin activation. Here we show that a key event in forming the active state of rhodopsin is proton uptake by Glu134 in the metarhodopsin II (MII) photoproduct. Site-directed mutants E134D and E134Q were studied by flash photolysis, where formation rates of their photoproducts and rates of pH change could be monitored simultaneously. Both mutants showed normal MII formation rates. However, E134D displayed a slowed rate of proton uptake and E134Q displayed a loss of light-induced uptake of two protons from the aqueous phase. Thus, Glu134 mediates light-dependent proton uptake by MII. We propose that receptor activation requires a light-induced conformational change that allows protonation of Glu134 and subsequent protonation of a second group. The strong conservation of Glu134 in G protein-coupled receptors implies a general requirement for a proton acceptor group at this position to allow light- or ligand-dependent receptor activation.

Modulation of rhodopsin function by properties of the membrane bilayer

A prevalent model for the function of rhodopsin centers on the metarhodopsin I (MI) to metarhodopsin II (MII) conformational transition as the triggering event for the visual process. Flash photolysis techniques enable one to determine the [MII]/[MI] ratio for rhodopsin in various recombinant membranes, and thus investigate the roles of the phospholipid head groups and the lipid acyl chains systematically. The results obtained to date clearly show that the p K for the acid-base MI-MII equilibrium of rhodopsin is modulated by the lipid environment. In bilayers of phosphatidylcholines the MI-MII equilibrium is shifted to the left; whereas in the native rod outer segment membranes it is shifted to the right, i.e., at neutral pH near physiological temperature. The lipid mixtures sufficient to yield full photochemical function of rhodopsin include a native-like head group composition, viz, comprising phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), in combination with polyunsaturated docosahexaenoic acid (DHA; 22:6ω3) chains. Yet such a native-like lipid mixture is not necessary for the MI-MII conformational transition of rhodopsin; one can substitute other lipid compositions having similar properties. The MI-MII transition is favored by relatively small head groups which produce a condensed bilayer surface, viz, a comparatively small interfacial area as in the case of PE, together with bulky acyl chains such as DHA which prefer a relatively large cross sectional area. The resulting force imbalance across the layer gives rise to a curvature elastic stress of the lipid/water interface, such that the lipid mixtures yielding native-like behavior form reverse hexagonal ( H II) phases at slightly higher temperatures. A relatively unstable membrane is needed; lipids tending to form the lamellar phase do not support full native-like photochemical function of rhodopsin. Thus chemically specific properties of the various lipids are not required, but rather average or material properties of the entire assembly, which may involve the curvature free energy of the membrane-lipid water interface. These findings reveal that the membrane lipid bilayer has a direct influence on the energetics of the conformational states of rhodopsin in visual excitation.

Structure and function in rhodopsin: replacement by alanine of cysteine residues 110 and 187, components of a conserved disulfide bond in rhodopsin, affects the light-activated metarhodopsin II state.

A disulfide bond that is evidently conserved in the guanine nucleotide-binding protein-coupled receptors is present in rhodopsin between Cys-110 and Cys-187. We have replaced these two cysteine residues by alanine residues and now report on the properties of the resulting rhodopsin mutants. The mutant protein C110A/C187A expressed in COS cells resembles wild-type rhodopsin in the ground state. It folds correctly to bind 11-cis-retinal and form the characteristic rhodopsin chromophore. It is inert to hydroxylamine in the dark, and its stability to dark thermal decay is reduced, relative to that of the wild type, by a delta delta G not equal to of only -2.9 kcal/mol. Further, the affinities of the mutant and wild-type rhodopsins to the antirhodopsin antibody rho4D2 are similar, both in the dark and in light. However, the metarhodopsin II (MII) and MIII photointermediates of the mutant are less stable than those formed by the wild-type rhodopsin. Although the initial rates of transducin activation are the same for both mutant and wild-type MII intermediates at 4 degrees C, at 15 degrees C the MII photointermediate in the mutant decays more than 20 times faster than in wild type. We conclude that the disulfide bond between Cys-110 and Cys-187 is a key component in determining the stability of the MII structure and its coupling to transducin activation.

Effect of ethanol on metarhodopsin II formation is potentiated by phospholipid polyunsaturation.

The role of phospholipids in modulating the effect of ethanol on membrane receptor activation was investigated by studying the extent of metarhodopsin II (MII) formation in vesicles formed POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and PDPC (1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine) and in native rod outer segment disk membranes as a function of ethanol concentration. The equilibrium concentration of MII, the G protein-activating form of photoactivated rhodopsin, was found to increase as a function of ethanol concentration in all three bilayers. Phospholipid composition had a marked effect on ethanol potency, with the presence of polyunsaturated phospholipid acyl chains increasing ethanol potency by 40%. The effects of ethanol on lipid acyl chain packing in POPC and PDPC were investigated using frequency domain anisotropy decay measurements of the fluorescent membrane probe 1,6-diphenyl-1,3,5-hexatriene. Enhanced formation of MII due to the presence of ethanol was correlated with the effects of ethanol on acyl chain packing properties. These findings support a phospholipid-mediated mechanism for the action of ethanol in modulating integral membrane receptor conformation.

Two different forms of metarhodopsin II: Schiff base deprotonation precedes proton uptake and signaling state.

Rhodopsin is a retinal protein and a G-protein-coupled receptor; it shares with both of these families the seven helix structure. To generate the G-interacting helix-loop conformation, generally identified with the 380-nm absorbing metarhodopsin II (MII) photoproduct, the retinal Schiff base bond to the apoprotein must be deprotonated. This occurs as a key event also in the related retinal proteins, sensory rhodopsins, and the proton pump bacteriorhodopsin. In MII, proton uptake from the aqueous phase must be involved as well, since its formation increases the pH of the aqueous medium and is accelerated under acidic conditions. In the native membrane, the pH effect matches MII formation kinetically, suggesting that intramolecular and aqueous protonation changes contribute in concert to the protein transformation. We show here, however, that proton uptake, as indicated by bromocresol purple, and Schiff base deprotonation (380-nm absorption change) show different kinetics when the protein is solubilized in suitable detergents. Our data are consistent with a two-step reaction:

Lipid headgroup and acyl chain composition modulate the MI-MII equilibrium of rhodopsin in recombinant membranes

A current paradigm for visual function centers on the metarhodopsin I (MI) to metarhodopsin II (MII) conformational transition as the trigger for an intracellular enzyme cascade leading to excitation of the retinal rod. We investigated the influences of the membrane lipid composition on this key triggering event in visual signal transduction using flash photolysis techniques. Bovine rhodopsin was combined with various phospholipids to form membrane recombinants in which the lipid acyl chain composition was held constant at that of egg phosphatidylcholine (PC), while the identity of the lipid headgroups was varied. The ratio of MII/MI produced in these recombinants by an actinic flash at 28 degrees C was studied as a function of pH. The results were compared to the photochemical function observed for rhodopsin in native retinal rod outer segment (ROS) membranes, in total native ROS lipid recombinants, and in dimyristoylphosphatidylcholine (DMPC) recombinants. In membrane recombinants incorporating lipids derived from egg PC, as well as in the total ROS lipids control and the native ROS disk membranes, MI and MII were found to coexist in a pH-dependent, acid-base equilibrium on the millisecond time scale. The recombinants of rhodopsin with egg PC, either alone or in combination with egg PC-derived phosphatidylethanolamine (PE) or phosphatidylserine (PS), exhibited substantially reduced photochemical activity at pH 7.0. However, all recombinants comprising phospholipids with unsaturated acyl chains were capable of full native-like MII production at pH 5.0, confirming previous results [Gibson, N.J.. & Brown, M.F. (1990) Biochem. Biophys. Res. Commun. 169, 1028-1034]. It follows that energetic constraints on the MI and MII states imposed by egg PC-derived acyl chains can be offset by increased activity of H+ ions. The data reveal that the major effect of the membrane lipid composition is to alter the apparent pK for the MI-MII conformational equilibrium of rhodopsin [Gibson, N.J., & Brown, M.F. (1991) Biochem. Biophys. Res. Commun. 176, 915-921]. Recombinants containing only phosphocholine headgroups exhibited the lowest apparent pK values, whereas the presence of either 50 mol % PE or 15 mol % PS increased the apparent pK. The inability to obtain full native-like function in recombinants having egg PC-derived chains and a native-like headgroup composition indicates a significant role of the polyunsaturated docosahexaenoic acid (DHA) chains (22:6 omega 3) of the native retinal rod membrane lipids. Temperature studies of the MI-MII transition enabled an investigation of lipid influences on the thermodynamic parameters of a membrane protein conformational change linked directly to function.(ABSTRACT TRUNCATED AT 400 WORDS)

Movement of the retinylidene Schiff base counterion in rhodopsin by one helix turn reverses the pH dependence of the metarhodopsin I to metarhodopsin II transition.

The environment of the retinylidene Schiff base in bovine rhodopsin has been studied by movement of its carboxylic acid counterion from position 113 to position 117 by site-specific mutagenesis. Replacement of the counterion at position 113 by a neutral amino acid residue has been shown to produce a lowering of the Schiff base acidity constant (pKa) from > 8.5 to about 6. The aim of the present work was to change the position of the counterion without causing a significant effect on the Schiff base pKa. A triple replacement mutant (Glu113-->Ala/Ala117-->Glu/Glu122-->Gln) was designed to move the position of the counterion by one helix turn in the third putative transmembrane helix (helix C). The mutant bound 11-cis-retinal to form a chromophore with a visible absorbance maximum (lambda max) of 490 nm which was independent of pH in the range of about 5-8.5. Upon illumination under conditions in which rhodopsin was converted to the active metarhodopsin II (MII) photoproduct, the mutant was converted to a metarhodopsin I (MI)-like species (lambda max = 475 nm). Furthermore, the effect of pH on the photobleaching behavior of the mutant was the reverse of that reported for rhodopsin. In the mutant, acidic pH favored the formation of the MI-like photoproduct, and basic pH favored the formation of an MII-like photoproduct (lambda max = 380 nm). The MII-like photoproduct of the mutant pigment was able to activate the guanine nucleotide-binding protein, transducin. We conclude that the Schiff base counterion in rhodopsin can be repositioned to form a pigment with an apparently unperturbed Schiff base pKa. Furthermore, a specific amino acid residue that acts as a Schiff base proton acceptor is not strictly required for photoconversion of rhodopsin to its active MII form.

Interaction of rhodopsin with the G‐protein, transducin

Rhodopsin, upon activation by light, transduces the photon signal by activation of the G-protein, transducin. The well-studied rhodopsin/transducin system serves as a model for the understanding of signal transduction by the large class of G-protein-coupled receptors. The interactive form of rhodopsin, R*, is conformationally similar or identical to rhodopsin's photolysis intermediate Metarhodopsin II (MII). Formation of MII requires deprotonation of rhodopsin's protonated Schiff base which appears to facilitate some opening of the rhodopsin structure. This allows a change in conformation at rhodopsin's cytoplasmic surface that provides binding sites for transducin. Rhodopsin's 2nd, 3rd and putative 4th cytoplasmic loops bind transducin at sites including transducin's 5 kDa carboxyl-terminal region. Site-specific mutagenesis of rhodopsin is being used to distinguish sites on rhodopsin's surface that are important in binding transducin from those that function in activating transducin. These observations are consistent with and extend studies on the action of other G-protein-coupled receptors and their interactions with their respective G proteins.

MEMBRANE LIPID INFLUENCES ON THE ENERGETICS OF THE METARHODOPSIN I and METARHODOPSIN II CONFORMATIONAL STATES OF RHODOPSIN PROBED BY FLASH PHOTOLYSIS*

We have investigated the relationship between rhodopsin photochemical function and the retinal rod outer segment (ROS) disk membrane lipid composition using flash photolysis techniques. Bovine rhodopsin was combined with various phospholipids to form recombinant membrane vesicles, in which the lipid acyl chain composition was maintained at that of egg phosphatidylcholine (PC), while the nature of the headgroups was varied. The ratio of metarhodopsin II (MII)/metarhodopsin I (MI) in these recombinants produced by an actinic flash was investigated as a function of pH, and compared with the photochemical activity observed for rhodopsin in native ROS membranes and dimyristoylphosphatidylcholine recombinants. In recombinants made with lipids derived from egg PC, as well as in native ROS membranes, MI and MII were found to be present in a pH-dependent, acid-base equilibrium on the millisecond timescale. The recombinants made with phospholipids containing unsaturated acyl chains were capable of full native-like MII production, but each demonstrated a titration curve with a different pK. In addition, some of the recombinants exhibited apparent deviations from the Henderson-Hasselbalch curve shape. The presence of either phosphatidylethanolamine (PE) or phosphatidylserine (PS) headgroups appeared to increase the amount of MII produced. This may result from alteration of the curvature free energy, in the case of PE, and from the influence of the membrane surface potential in the case of PS. An investigation of the effects of temperature on the MI-MII transition in native ROS membranes and the recombinants was also carried out.(ABSTRACT TRUNCATED AT 250 WORDS)

Displacement of rhodopsin by GDP from three-loop interaction with transducin depends critically on the diphosphate beta-position.

We have studied the effect of GDP and its analog guanyl-5'-yl thiophosphate (GDP beta S) on the interaction between rhodopsin and transducin (Gt). Stabilization of the light-induced active intermediate, metarhodopsin II (MII), by bound Gt (extra-MII effect) monitored the catalytic interaction between the proteins. Extra-MII can be completely abolished by GDP, with a half-suppression at 10 microM under the conditions (4 degrees C, pH 8, 7.5 nM photoactivated rhodopsin). The effect of GDP did not depend on divalent cations, in contrast to GTP-induced dissociation of the complex. The GDP analog GDP beta S did not affect extra-MII although it binds to the MII-Gt complex with only three times lower affinity (reversal of the GDP effect by GDP beta S). However, GDP beta S enhanced considerably the efficiency of synthetic rhodopsin peptide competition against the formation of extra-MII. GDP and GDP beta S slow the Gt activation rate (monitored by kinetic light scattering), with the same relative efficiencies. We therefore assume that GDP, GDP beta S, and GTP bind at the same site. We discuss a generalized induced fit mechanism, where MII induces opening of the Gt nucleotide site and release of GDP which in turn is obligatory to establish the MII-stabilizing rhodopsin-Gt three-loop interaction (König, B., Arendt, A., McDowell, J.H., Kahlert, M., Hargrave, P.A., and Hofmann, K.P. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6878-6882). The GDP beta S/GDP difference is discussed in terms of bound GDP disturbing the interaction with two and GDP beta S with only one of the rhodopsin binding sites. Mechanistically, our results indicate a critical role of the beta-phosphate interaction with the nucleotide binding site in the GDP-induced transformation of Gt.

Influence of pH on the MI-MII equilibrium of rhodopsin in recombinant membranes

Rhodopsin in native rod membranes and incorporated into egg phosphatidylcholine (egg PC) vesicles was studied at pH 5 and 7 at 28°C. Rhodopsin function, as monitored by the formation of metarhodopsin II (MII) from metarhodopsin I (MI) following an actinic flash, was found to be largely blocked in egg PC vesicles at pH 7. When the pH was lowered to 5, however, rhodopsin showed essentially equal activity in both native and egg PC membranes. This activity exceeded that found for rhodopsin in native membranes at pH 7. Phospholipid composition is thus shown to directly affect the MI ⇌ MII equilibrium, which in turn is linked to visual function.

Photoactivation of rhodopsin and interaction with transducin in detergent micelles: Effect of ‘doping’ with steroid molecules

On detergent-solubilised bovine rhodopsin, we have studied the formation of the active photoproduct, metarhodopsin II (MII), and its interaction with the rod G-protein, transducin (G t). The measured rate of flash-induced MII formation decreases by a factor of 300 from n-dodecyl-β-D-maltoside ( k = 4 × 10 3 at 18°C, pH 6.5), over (3-(lauroyloxy)propyl)-phosphorylcholine (deoxylysolecithin), n-octyl-β-D-glucopyranoside, sodium cholate to Chapso. For the two last agents. MII formation is similarly slow as in the native disc membrane; however, the micellar system does not display the very large decrease of the rate with lowering temperature, as is characteristic for the membrane. This points to entropic factors determining the rate in the micellar systems. An admixture of rigid steroid molecules (11-deoxycorticosterone) to lysolecithin micelles (‘doped micelles’) slows MII formation and shifts the MI/MII equilibrium to values typical for detergents of rigid structure. The observation gives further support to the surface free energy concept of MII formation outlined in previous studies. The free adjustment of the MI/MII equilibrium in these doped micelles allows G t-induced formation of extra-MII to be measured, providing a convenient monitor of rhodopsin-G t interaction in solution. Rhodopsin; Metarhodopsin II; Transducin; G-protein; (Detergent micelle)

Kinetics, binding constant, and activation energy of the 48-kDa protein-rhodopsin complex by extra-metarhodopsin II.

We have found that the 48-kDa protein (or S-antigen 48k) of the rod photoreceptor enhances the light-induced formation of the photoproduct metarhodopsin II (MII) from prephosphorylated rhodopsin. The effect is analogous to the known enhancement of MII (extra-MII) that results from selective interaction of MII with G-protein. We have determined some parameters of the MII-48k interaction by measuring the extra-MII absorption change induced by the 48-kDa protein. The amplitude saturation yields a dissociation constant for the MII-48k complex on the order of 50 nM. At the technical limit of these measurements, 13.7 degrees C and 12 microM 48-kDa protein, we find a rate of 2.3 s-1 for formation of the 48k-MII complex. Extrapolation of these values to cellular conditions yields an occupation time of phosphorylated MII by 48k less than 200 ms. This is short compared to estimated rates of phosphorylation. The temperature dependence of the MII-48k formation rate is very high (Q10 for 5 degrees C/15 degrees C = 9-10). The related Arrhenius activation energy (165 kJ mol-1) is correspondingly high and indicates a considerable transient chemical change during the binding process.

Can metarhodopsin I activate rod outer segment phosphodiesterase?

The rod photoreceptors of vertebrate retinas contain a cGMP phosphodiesterase (PDE) that is activated by light. The light is absorbed by rhodopsin that activates an intermediate GTP-binding protein; this species then activates the PDE. Photo-excited rhodopsin passes through a series of transient states, and the purpose of this study is to identify the earliest state that interacts with the GTP-binding protein and thus activate the PDE. The majority of evidence points to this state being metarhodopsin II (MII), but PDE activation is seen at low temperatures where the rhodopsin reaction sequence is not expected to pass beyond the metarhodopsin I (MI) stage. Light thresholds for PDE activation have been determined under conditions where little MII is generated, and these are compared with the concentration of MII. The conclusion is that for a criterion threshold of PDE activity, the MII concentration is constant, irrespective of the amount of MI present, which suggests that MI cannot activate the PDE system.

Deoxylysolecithin and a new biphenyl detergent as solubilizing agents for bovine rhodopsin. Functional test by formation of metarhodopsin II and binding of G-protein.

The protein-detergent interaction in rhodopsin-detergent micelles has been investigated by using formation of metarhodopsin II (MII) as a monitor. Two detergents of different structural rigidity have been applied. One of them is [3-(lauroyloxy)propyl]phosphorylcholine, which has a high conformational flexibility in its hydrophobic moiety like most of the known detergents for rhodopsin. This deoxylysolecithin was originally designed as a detergent for membrane proteins by Weltzien [Weltzien, H. U. (1979) Biochim. Biophys. Acta 559, 259-287]. The other detergent, which is highly rigid in its hydrophobic part, has been developed for this study. It consists of a biphenyl derivative and a hydrophilic octaethylene oxide group. Both the formation kinetics of MII and the position of its equilibrium with its tautomeric form, metarhodopsin I (MI), strongly differed in the deoxylysolecithin and biphenyl detergent. Deoxylysolecithin caused very fast MII formation and shifted the equilibrium strongly to MII, like other detergents with alkyl chains as the hydrophobic part. In the biphenyl detergent, however, formation of MII was slow and the MI/MII equilibrium similar to that in the native system. For rhodopsin reconstituted in lipid bilayers, normal MII formation requires a well-adjusted fluidity of the hydrocarbon environment of the protein [Baldwin, P. A., & Hubbell, W. L. (1984) Biochemistry 24, 2633-2639], which was explained by an appropriate interfacial pressure at the protein-lipid interface. Extension of this concept would indicate that in the micellar core a degree of fluidity comparable to that of the disk membrane is just achieved with the highly rigid biphenyl structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction between photoexcited rhodopsin and peripheral enzymes in frog retinal rods. Influence on the postmetarhodopsin II decay and phosphorylation rate of rhodopsin.

The major peripheral and soluble proteins in frog rod outer segment preparations, and their interactions with photoexcited rhodopsin, have been compared to those in cattle rod outer segments and found to be similar in both systems. In particular the GTP-binding protein (G) has the same subunit composition, the same abundance relative to rhodopsin (1/10) and it undergoes the same light and nucleotide-dependent interactions with rhodopsin in both preparations. Previous work on cattle rod outer segments has shown that photoexcited rhodopsin (R*), in a state identified with metarhodopsin II, associates with the G protein as a first step to the light-activated GDP/GTP exchange on G. The complex R*-G is stable in absence of GTP, but is rapidly dissociated by GTP owing to the GDP/GTP exchange reaction. Low bleaching extents (less than 10% R*) in absence of GTP therefore create predominantly R*-G complexes, whereas bleaching in presence of GTP creates free R*. We report here that, under conditions of complexed R*, two reactions of R* in frog rod outer segments are highly perturbed as compared to free R*: (a) the spectral decay of metarhodopsin II (MII) into later photoproducts, and (b) the phosphorylation of R* by an ATP-dependent protein kinase. a) The spectral measurements have been performed using linear dichroism on oriented frog rod outer segments; this technique allows discrimination between MII and later photoproducts absorbing at the same wavelength. Association of R* with G leads to a strong reduction of the amount of MIII formed and to an acceleration of the decay of MIII. Furthermore, MII is significantly stabilized, in agreement with the hypothesis that MII is the intermediate which binds to G. b) The phosphorylation of R* is strongly inhibited under conditions of R*-G complex formation as compared to free R*. Interferences between reactions at the three sites involved in R* are discussed: the retinal binding site in the hydrophobic core is sensitive to the presence of GTP-binding protein at its binding site on the cytoplasmic surface of R*; the kinase and the GTP-binding protein compete for access to their respective binding sites, both located on the surface of R*. We also observed a slow and nucleotide-dependent light-induced binding of a protein of molecular weight 50 000, which we consider as the equivalent of the 48 000 Mr light-dependent protein previously identified in cattle rod outer segments.

Complex formation between metarhodopsin II and GTP-binding protein in bovine protoreceptor membranes leads to a shift of the photoproduct equilibrium

FEBS LettersVolume 143, Issue 1 p. 29-34 Full-length articleFree Access Complex formation between metarhodopsin II and GTP-binding protein in bovine protoreceptor membranes leads to a shift of the photoproduct equilibrium D. Emeis, D. Emeis Institut für Biophysik und Strahlenbiologie der Universität Freiburg in Breisgau, Albertstrasse 23, 7800 Freiburg i. Br. FRGSearch for more papers by this authorH. Kühn, H. Kühn Institut für Neurobiologie der Kernforschungsanlage, Jülich GmbH, PO Box 1913, 5170 Jülich, FRGSearch for more papers by this authorJ. Reichert, J. Reichert Institut für Biophysik und Strahlenbiologie der Universität Freiburg in Breisgau, Albertstrasse 23, 7800 Freiburg i. Br. FRGSearch for more papers by this authorK.P. Hofmann, K.P. Hofmann Institut für Biophysik und Strahlenbiologie der Universität Freiburg in Breisgau, Albertstrasse 23, 7800 Freiburg i. Br. FRGSearch for more papers by this author D. Emeis, D. Emeis Institut für Biophysik und Strahlenbiologie der Universität Freiburg in Breisgau, Albertstrasse 23, 7800 Freiburg i. Br. FRGSearch for more papers by this authorH. Kühn, H. Kühn Institut für Neurobiologie der Kernforschungsanlage, Jülich GmbH, PO Box 1913, 5170 Jülich, FRGSearch for more papers by this authorJ. Reichert, J. Reichert Institut für Biophysik und Strahlenbiologie der Universität Freiburg in Breisgau, Albertstrasse 23, 7800 Freiburg i. Br. FRGSearch for more papers by this authorK.P. Hofmann, K.P. Hofmann Institut für Biophysik und Strahlenbiologie der Universität Freiburg in Breisgau, Albertstrasse 23, 7800 Freiburg i. Br. FRGSearch for more papers by this author First published: June 21, 1982 https://doi.org/10.1016/0014-5793(82)80266-4Citations: 214AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume143, Issue1June 21, 1982Pages 29-34 ReferencesRelatedInformation

52] Bleaching intermediate kinetics of rhodopsin, metarhodopsin I, and metarhodopsin II

This chapter discusses the bleaching intermediate kinetics of rhodopsin, metarhodopsin I, and metarhodopsin II. When cattle rhodopsin absorbs light under near-physiological conditions, it is rapidly converted to metarhodopsin I (MI), which, in turn, decays to metarhodopsin II (MII). From the very earliest determination of the rate of MII accumulation, it has been reported that the kinetics are not simple first order. Rather, the rate has been accurately described by the sum of two exponentials. It appears that one fraction of the rhodopsin, which absorbs light and produces MII, does so at a slightly faster rate than the other. The ratio of rate constants is always about 5:1, but the relative amounts of the “fast-appearing MII” and “slow-appearing MII” depend on many experimental parameters. In terms of specific effects on MI→MII kinetics, temperature is a key parameter. The kinetic behavior of the transition of meta I to meta II that one observes is strongly dependent on two variables—namely, temperature and solvating environment.