Biomedical subjects

T G Ebrey

Publications and source records attributed to T G Ebrey.

At least 37 records · Page 2Linked to original sources

PubMed1997

Charge movements in the 13-cis photocycles of the bacteriorhodopsin mutants R82K and R82Q.

We have examined light-induced currents in oriented membranes of the bacteriorhodopsin mutants R82K and R82Q. Our results suggest that two photocurrent components found in R82K, with 30 and 300 microseconds lifetimes, are due to the photocycle of the 13-cis rather than the all-trans form of the pigment. We investigated the pH dependence of these components and their correspondence to absorbance changes at 660 nm characteristic of photointermediates of the 13-cis cycle. The presence of a D2O effect suggests that the charge motions producing these photocurrents are related to proton or protonated amino acid movement within the molecule. The current amplitudes depend on the protonation states of at least two residues, D85 and (probably) E204. In R82Q, a 10 microseconds photocurrent is observed that also depends on the protonation state of D85 and is similar to the 30 microseconds current in R82K. We attempt to explain these currents in terms of a model for interacting residues in the extracellular half of the bacteriorhodopsin channel.

Adaptation, Physiological↗

PubMed1996

Effects of arginine-82 on the interactions of internal water molecules in bacteriorhodopsin.

Arg82, one of the residues near the protonated Schiff base of bacteriorhodopsin, facilitates proton release to the medium during the L-to-M reaction of the photocycle, but retards the rate of proton transfer from the Schiff base to Asp85. In order to understand the role of Arg82 in these processes, the structural changes upon formation of the M intermediate were studied by Fourier transform infrared spectroscopy of the hydrated films of Arg82 mutants at pH 9.5. The negative band at 1700 cm-1 in the BR --> M spectrum due to the deprotonation of Glu204 was absent when Arg82 was replaced with alanine (R82A), but present with small amplitude when residue 82 was a glutamine (R82Q), or a lysine (R82K), with a shift to 1696 cm-1. The O-H stretch of water at 3643 cm-1 is shifted toward a lower frequency in R82Q, R82K, and R82A in the unphotolyzed state. However, R82Q retains a fraction of the unshifted band. Another O-H stretch is prominent in R82Q around 3625 cm-1 but absent in R82A and probably in R82K. In parallel, R82Q retains a fraction of the slow component of the formation of the M intermediate, which is almost completely absent in R82K and R82A. These results, along with previous data for the mutants of Glu204, suggest that the guanidium group of Arg82 influences the H-bonding of water molecules located close to Asp85 and Arg82-Glu204 regions, and the rate of proton transfer from the Schiff base to Asp85. The amide group of Gln82 can substitute for it but weakly.

Arginine↗

PubMed1996

Arginine-82 regulates the pKa of the group responsible for the light-driven proton release in bacteriorhodopsin.

In wild-type bacteriorhodopsin light-induced proton release occurs before uptake at neutral pH. In contrast, in mutants in which R82 is replaced by a neutral residue (as in R82A and R82Q), only a small fraction of the protons is released before proton uptake at neutral pH; the major fraction is released after uptake. In R82Q the relative amounts of the two types of proton release, "early" (preceding proton uptake) and "late" (following proton uptake), are pH dependent. The main conclusions are that 1) R82 is not the normal light-driven proton release group; early proton release can be observed in the R82Q mutant at higher pH values, suggesting that the proton release group has not been eliminated. 2) R82 affects the pKa of the proton release group both in the unphotolyzed state of the pigment and during the photocycle. In the wild type (in 150 mM salt) the pKa of this group decreases from approximately 9.5 in the unphotolyzed pigment to approximately 5.8 in the M intermediate, leading to early proton release at neutral pH. In the R82 mutants the respective values of pKa of the proton release group in the unphotolyzed pigment and in M are approximately 8 and 7.5 in R82Q (in 1 M salt) and approximately 8 and 6.5 in R82K (in 150 mM KCl). Thus in R82Q the pKa of the proton release group does not decrease enough in the photocycle to allow early proton release from this group at neutral pH. 3) Early proton release in R82Q can be detected as a photocurrent signal that is kinetically distinct from those photocurrents that are due to proton movements from the Schiff base to D85 during M formation and from D96 to the Schiff base during the M-->N transition. 4) In R82Q, at neutral pH, proton uptake from the medium occurs during the formation of O. The proton is released during the O-->bacteriorhodopsin transition, probably from D85 because the normal proton release group cannot deprotonate at this pH. 5) The time constant of early proton release is increased from 85 microseconds in the wild type to 1 ms in R82Q (in 150 mM salt). This can be directly attributed to the increase in the pKa of the proton release group and also explains the uncoupling of proton release from M formation. 6) In the E204Q mutant only late proton release is observed at both neutral and alkaline pH, consistent with the idea that E204 is the proton release group. The proton release is concurrent with the O-->bacteriorhodopsin transition, as in R82Q at neutral pH.

Amino Acid Sequence↗

PubMed1996

Evidence that aspartate-85 has a higher pK(a) in all-trans than in 13-cisbacteriorhodopsin.

Three experimental observations indicate that the pK(a) of the purple-to-blue transition (the pK(a) of Asp-85) is higher for all-trans-bR(1) than for 13-cis-bR. First, light adaptation of bacteriorhodopsin (bR) at pHs near the pK(a) of Asp-85 causes an increase in the fraction of the blue membrane present. This transformation is reversible in the dark. Second, the pK(a) of the purple-to-blue transition in the dark is lower than that in the light-adapted bR (pK(a)(DA) = 3.5, pK(a)(LA) = 3.8 in 10 microM K(2)SO(4)). Third, the equilibrium fractions of 13-cis and all-trans isomers are pH dependent; the fraction of all-trans-bR increases upon formation of the blue membrane. Based on the conclusion that thermal all-trans <=> 13-cis isomerization occurs in the blue membrane rather than in the purple, we have developed a simple model that accounts for all three observations. From the fit of experimental data we estimate that the pK(a) of Asp-85 in 13-cis-bR is 0.5 +/- 0.1 pK(a) unit less than the pK(a) of all-trans-bR. Thus in 10 microM K(2)SO(4), pK(a)(c) = 3.3, whereas pK(a)(t) = 3.8.

Aspartic Acid↗

PubMed1996

Titration of aspartate-85 in bacteriorhodopsin: what it says about chromophore isomerization and proton release.

Titration of Asp-85, the proton acceptor and part of the counterion in bacteriorhodopsin, over a wide pH range (2-11) leads us to the following conclusions: 1) Asp-85 has a complex titration curve with two values of pKa; in addition to a main transition with pKa = 2.6 it shows a second inflection point at high pH (pKa = 9.7 in 150-mM KCl). This complex titration behavior of Asp-85 is explained by interaction of Asp-85 with an ionizable residue X'. As follows from the fit of the titration curve of Asp-85, deprotonation of X' increases the proton affinity of Asp-85 by shifting its pKa from 2.6 to 7.5. Conversely, protonation of Asp-85 decreases the pKa of X' by 4.9 units, from 9.7 to 4.8. The interaction between Asp-85 and X' has important implications for the mechanism of proton transfer. In the photocycle after the formation of M intermediate (and protonation of Asp-85) the group X' should release a proton. This deprotonated state of X' would stabilize the protonated state of Asp-85.2) Thermal isomerization of the chromophore (dark adaptation) occurs on transient protonation of Asp-85 and formation of the blue membrane. The latter conclusion is based on the observation that the rate constant of dark adaptation is directly proportional to the fraction of blue membrane (in which Asp-85 is protonated) between pH 2 and 11. The rate constant of isomerization is at least 10(4) times faster in the blue membrane than in the purple membrane. The protonated state of Asp-85 probably is important for the catalysis not only of all-trans <=> 13-cis thermal isomerization during dark adaptation but also of the reisomerization of the chromophore from 13-cis to all-trans configuration during N-->O-->bR transition in the photocycle. This would explain why Asp-85 stays protonated in the N and O intermediates.

Aspartic Acid↗

PubMed1995

The two pKa's of aspartate-85 and control of thermal isomerization and proton release in the arginine-82 to lysine mutant of bacteriorhodopsin.

To explore the role of Arg82 in the catalysis of proton transfer in bacteriorhodopsin, we replaced Arg82 with Lys, which is also positively charged at neutral pH but has an intrinsic pKa of about 1.7 pH units lower than that of Arg. In the R82K mutant expressed in Halobacterium salinarium, we found the following: (1) The pKa of the purple-to-blue transition at low pH (which reflects the pKa of Asp85) is 3.6 +/- 0.1. At high pH a second inflection in the blue-to-purple transition with pKa = 8.0 is found. The complex titration behavior of Asp85 indicates that the pKa of Asp85 depends on the protonation state of another amino acid residue, X', which has a pKa = 8.0 in R82K. The fit of the experimental data to a model of two interacting residues shows that deprotonation of X' at high pH causes a shift in the pKa of Asp85 from 3.7 to 6.0. In turn, protonation of Asp85 decreases the pKa of X' by 2.3 pH units. This suggests that X' can release a proton upon formation of the M intermediate and the concomitant protonation of Asp85 in the photocycle. (2) The rate constant of dark adaptation, kda, is proportional to the fraction of blue membrane between pH 2 and 10, indicating that thermal isomerization proceeds through the transient protonation of Asp85. The pH dependence of kda shows that two groups with pKal = 3.9 and pKa2 = 8.0 control the rate of dark adaptation in R82K. The 1.7 pH unit shift in pKa2 in R82K compared to the wild type (WT) (pKa2 = 9.7) supports the hypothesis that X' is Arg82 in WT and Lys82 in R82K (or at least that these groups are the principal part of a cluster of residues that constitute X'). (3) Under steady state illumination, the efficiency of proton transport in R82K incorporated in phosphatidylcholine vesicles is at least 40% of that in the WT. A flash-induced transient signal of the pH-sensitive dye pyranine is similar to that in the WT (proton release precedes uptake), but the amplitude is small in R82K (about 15% of that found in the WT), indicating that only a small fraction of protons is released fast in R82K. This supports the suggestions that Arg82 is associated with the proton release pathway (acts as a proton release group or part of a proton release complex) and that Lys cannot efficiently substitute for Arg in this process.(ABSTRACT TRUNCATED AT 400 WORDS)

Arginine↗

PubMed1995

Chloride effect on the early photolysis intermediates of a gecko cone-type visual pigment.

Nanosecond laser photolysis measurements were conducted on the cone-type visual pigment P521 in digitonin extracts of Tokay gecko (Gekko gekko) retina containing physiological chloride ion levels and also on samples which had been chloride depleted or which contained high levels (4 M) of chloride. Absorbance difference spectra were recorded at a sequence of time delays from 30 ns to 60 microseconds following excitation with a pulse of either 532- or 477-nm actinic light. Global analysis showed the kinetic decay data for gecko pigment P521 to be best fit by two exponential processes under all chloride conditions. The initial photoproduct detected had a broad spectrum characteristic of an equilibrated mixture of a Batho P521 intermediate with its blue-shifted intermediate (BSI P521) decay product. The first exponential process was assigned to the decay of this mixture to the Lumi P521 intermediate. The second exponential process was identified as the decay of Lumi P521 to Meta I P521. The initial photoproduct's spectrum exhibited a strong dependence on chloride concentration, indicating that chloride affects the composition of the equilibrated mixture of Batho P521 and BSI P521. These results suggest that the affinity for chloride is reduced approximately 5-fold in the Batho P521 intermediate and approximately 50-fold in the BSI P521 intermediate. Chloride concentration also affects the apparent decay rate of the equilibrated mixture. When the apparent decay rate is corrected for the composition of the equilibrated mixture, a relatively invariant microscopic rate constant is obtained for BSI decay (k = 1/55 ns-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Effects of substitution of tyrosine 57 with asparagine and phenylalanine on the properties of bacteriorhodopsin.

Tyrosine 57 is one of the residues present in the retinal binding pocket and is conserved in all the halophilic retinal proteins. We have studied mutants of bacteriorhodopsin, expressed in Halobacterium salinarium, in which tyrosine 57 is replaced by an asparagine (Y57N) or phenylalanine (Y57F). In Y57N the photocycle proceeds only up to the L intermediate; no M is formed at neutral pH. The lifetime of L intermediate is extremely long, ca. 500 ms. Proton release is severely affected in both the mutants which suggests that Y57 is associated with the proton release pathway. By comparing the pH-induced absorption changes in the UV in Y57N and Y57F with those in the wild-type (WT), we determined that the pKa of Y57 is 10.2. In Y57F, which shows M formation, the rate constant of the L-->M transition is pH dependent (pKa 8.7) suggesting that Y57 is probably not the residue that normally controls the transition into the alkaline photocycle. Y57 is either part of the counterion complex or in close proximity to D85 since its mutation influences the pKa of Asp85. In Y57F the pKa of D85 is approximately 4.9 (compared to approximately 2.9 in the WT). The Y57N mutant shows two pKa's in the purple to blue transition, approximately 3.8 and < 1. In the presence of hydroxylamine, at neutral pH, Y57N is stable in the dark but bleaches very rapidly upon illumination compared to the WT. Since the lifetime of L intermediate is long in Y57N, we suggest that the Schiff base becomes accessible to hydroxylamine in this state.

Asparagine↗

PubMed1994

Probing of the retinal binding site of bacteriorhodopsin by affinity labeling.

The position of the chromophore within bacteriorhodopsin has been identified by cross-linking a cysteine group, introduced by site-specific mutagenesis, with a chromophore suitably derivatized with an active leaving group. Since bacteriorhodopsin has no cysteines, a site-specific cysteine mutant will contain only one free sulfhydryl group capable of reacting with the retinal analog. Met118, Thr121, and Ser141 were selected to be mutated to cysteine. No pigment absorbing in the visible region was obtained for the Ser141Cys mutant. The Met118Cys and Thr121Cys mutants have similar absorption maxima, proton pumping efficiencies and photocycles to those of the wild-type pigment. 4-Bromoretinal, in which the reactive allylic halide readily undergoes nucleophilic displacement, was used as the reactive chromophore. Pigments were obtained on reaction of all-trans-4-bromoretinal with the apoproteins of Met118Cys, Thr121Cys, and wild-type bacteriorhodopsin (lambda max = 464-470 nm). Analysis of the denatured pigments on SDS-polyacrylamide gels showed incorporation of tritiated chromophore into the Met118Cys mutant but not into the wild-type or Th4121Cys pigments. Met118Cys apoprotein which was preincubated with the cysteine-specific reagent N-ethylmaleimide formed a pigment with 4-bromoretinal but no cross-linking was observed, providing evidence that the cross-linking of the chromophore is to the cysteine at 118. We conclude that Met118 is positioned in the chromophore binding pocket, proximal to the C-4 position of cyclohexyl ring of retinal.

Affinity Labels↗

PubMed1994

The pKa of the protonated Schiff bases of gecko cone and octopus visual pigments.

A visual pigment is composed of retinal bound to its apoprotein by a protonated Schiff base linkage. Light isomerizes the chromophore and eventually causes the deprotonation of this Schiff base linkage at the meta II stage of the bleaching cycle. The meta II intermediate of the visual pigment is the active form of the pigment that binds to and activates the G protein transducin, starting the visual cascade. The deprotonation of the Schiff base is mandatory for the formation of meta II intermediate. We studied the proton binding affinity, pKa, of the Schiff base of both octopus rhodopsin and the gecko cone pigment P521 by spectral titration. Several fluorinated retinal analogs have strong electron withdrawing character around the Schiff base region and lower the Schiff base pKa in model compounds. We regenerated octopus and gecko visual pigments with these fluorinated and other retinal analogs. Experiments on these artificial pigments showed that the spectral changes seen upon raising the pH indeed reflected the pKa of the Schiff base and not the denaturation of the pigment or the deprotonation of some other group in the pigment. The Schiff base pKa is 10.4 for octopus rhodopsin and 9.9 for the gecko cone pigment. We also showed that although the removal of Cl- ions causes considerable blue-shift in the gecko cone pigment P521, it affects the Schiff base pKa very little, indicating that the lambda max of visual pigment and its Schiff base pKa are not tightly coupled.

Animals↗

PubMed1994

Improved large-scale purification of transducin, and its alpha and beta gamma subunits from frozen retinas.

The transducin heterotrimer and its alpha- and beta gamma-subunits have been purified from frozen bovine rod outer segments by modifying existing procedures. The methods described here are relatively simple and fast. The yield (ca. 8 mgs/100 retinas) and purity of the transducin heterotrimer and subunits from frozen retinas is equal to or larger than those previously obtained from fresh or frozen retinas.

Animals↗

PubMed1994

Azidotetrafluorophenyl retinal analogue: synthesis and bacteriorhodopsin pigment formation.

The retinal derivative, all-trans-9-(4-azido-2,3,5,6-tetrafluorophenyl)-3,7- dimethyl-2,4,6,8-nonatetraenal, was synthesized by two routes as a potential photoactivatable cross-linking agent for studies in bacteriorhodopsin (BR) of the chromophore interaction with its apoprotein. The retinal analogue formed a stable, moderately functional BR pigment confirming that the ring cavity of the retinal binding site has a significant tolerance for derivatization on that portion of the molecule. Attempts to cross-link the azido chromophore to the protein by photoactivation were unsuccessful. The electron delocalization effect of the conjugated polyene side chain of the retinal appears to interfere with the formation or reactivity of the nitrene intermediate to the extent that photoactivated cross-linking is not achieved. These results demonstrate a limitation to the use of fluorinated aryl azides as photoaffinity reagents.

Bacteriorhodopsins↗

PubMed1994

pH dependence of the absorption spectra and photochemical transformations of the archaerhodopsins.

Two strains of archaebacteria have been found to contain light-driven proton pumping pigments analogous to bacteriorhodopsin (bR) in Halobacterium salinarium. These proteins are called archaerhodopsin-1 (aR-1) and archaerhodopsin-2 (aR-2). Their high degree of sequence identity with bR within the putative proton channel enables us to draw some conclusions about the roles of regions where differences in amino acids exist, and in particular the surface residues, on the structure and function of retinal-based proton pumps. We have characterized the spectral and photochemical properties of these two proteins and compared them to the corresponding properties of bR. While there are some differences in absorbance maxima and kinetics of the photocycle, most of the properties of aR-1 and aR-2 are similar to those of bR. The most striking differences of these proteins with bR are the lack of an alkaline-induced red-shifted absorption species and a dramatic (apparent) decrease in the light-induced transient proton release. In membrane sheet suspensions of aR-1 at 0.15 M KCl, the order of proton release and uptake appears opposite that of bR, in which proton release precedes uptake. The nature of this behavior appears to be due to differences in the amino acid sequence at the surfaces of the proteins. In particular, the residue corresponding to the lysine at position 129 of the extracellular loop region of bR is a histidine in aR-1 and could regulate the efficient release of protons into solution in bR.

Archaeal Proteins↗

PubMed1993

Metarhodopsin intermediates of the gecko cone pigment P521.

Cone visual pigments are responsible for color vision. Using low-temperature spectroscopy and linear/logarithmic time scale flash photolysis, we studied the photochemistry of the cone visual pigment P521 from the lizard Gekko gekko. We found both meta I and meta II intermediates in this cone pigment; meta I absorbs at 480 nm, and meta II absorbs at ca. 380 nm. The formation of meta I is a fast process with a lifetime of 2.8 microseconds for the slow component (pH 6.70, 8.0 degrees C, 2% digitonin) compared to 2.1 microseconds for bovine rhodopsin under these conditions. The formation of meta II does not have single-exponential kinetics but can be better characterized by fast and slow components. High pH favors faster kinetics of meta II formation, but less is formed. The amount of meta II formed has a pKa of 8.7. The fast component of the meta II formation seems to have a somewhat lower pKa (e.g., 6.4). Temperature also affects meta II formation, with high temperature favoring a faster rate and larger amounts. The higher pKa of the meta I to meta II transition in gecko P521 compared to a rod pigment like bovine rhodopsin (pKa = 6.4) probably is due to a cysteine residue at position 211 in gecko rather than a histidine residue in bovine rhodopsin.

Animals↗

PubMed1993

Effect of the arginine-82 to alanine mutation in bacteriorhodopsin on dark adaptation, proton release, and the photochemical cycle.

The pH dependence of the rate constant of dark adaptation (thermal isomerization from all-trans- to 13-cis-bR) drastically changes when Arg82 of bacteriorhodopsin is replaced by an alanine. In the wild type (WT) the rate decreases sharply between pH 2.5 and pH 5. In R82A the sharp decrease is shifted to pH > 7. This correlates with the shift in the pK of the purple-to-blue transition from pH 2.6 in the wild type to pH 7.2 in the mutant (in 150 mM KCl). We propose that the same group that controls the purple-to-blue transition, namely, Asp85, catalyzes dark adaptation. The rate of dark adaptation in the R82A mutant is proportional to the fraction of protonated Asp85, indicating that dark adaptation occurs when Asp85 is transiently protonated. Thermal isomerization is at least 2 x 10(3) times more likely when Asp85 is protonated (blue membrane) than when it is deprotonated (purple membrane). The pH dependence of dark adaptation in the WT can be explained by a model in which the rate of dark adaptation in the WT is also proportional to the fraction of protonated Asp85 and that the pK of Asp85 depends on some other group, X, which deprotonates (or moves away from Asp85) with pK9 and causes the shift in the pK of Asp85 from 2.6 to 7.2. The quantum yield of light adaptation is at least an order of magnitude less in R82A as compared to the WT. The rise time of M formation is very fast in R82A and, unlike the WT, pH independent (1 microsecond versus 85 and 6 microseconds in the WT at pH 7 and 10, respectively). The activation energy of the L to M transition is 6.9 kcal/mol versus 13.5 kcal/mol in the WT. Thus the loss of a positive charge in the active site greatly increases the rate of light-induced deprotonation of the Schiff base. In the R82A mutant, the M decay at pH > 8.8 is much faster than the recovery of initial bR, which suggests a decrease in the rate of back-reaction from N to M. In a suspension of R82A membranes the rate of proton release as measured by the pH-sensitive dye pyranine is delayed by at least 20-fold (in 2 M KCl), while the uptake of protons did not change much (12 ms in the WT versus 8 ms in R82A).(ABSTRACT TRUNCATED AT 400 WORDS)

Alanine↗

PubMed1993

pH dependence of light-induced proton release by bacteriorhodopsin.

We have measured the current generated by light-activated proton release from bacteriorhodopsin into solution as a function of both pH and ionic strength. We find that proton release into solution decreases with increasing pH with an intrinsic pKa of 8.2 +/- 0.2. This pH dependence indicates that the deprotonation of a certain group inhibits or abolishes proton release. Under physiological conditions, this group either releases a proton directly into solution or interacts with the site of proton release. The most immediate candidates for this protonatable species are tyrosine-57, tyrosine-185, arginine-82, and water; acting individually or cooperatively. The salt dependence of the apparent pKa of this group also allows us to calculate the surface charge density of about -5 charges per bacteriorhodopsin, compatible with previous estimates.

Bacteriorhodopsins↗

PubMed1993

Decreased energy requirement of toad retina during light adaptation as demonstrated by 31P nuclear magnetic resonance.

1. The effect of light and dark adaptation on the levels of phosphorus metabolites (nucleotide di- and triphosphates, phosphocreatine, pyridine nucleotide, inorganic phosphate, phosphodiesters, phosphomonoesters, and uridine diphosphate-glucose) in the toad (Bufo marinus) retina and retinal extracts was studied by 31P nuclear magnetic resonance (NMR) spectroscopy. 2. Spectra were acquired using an NMR probe specifically designed for superfusion and illumination of a single retina. Retinae were maintained at a steady state for up to 10 h in an electrolyte solution containing 10 mM Hepes buffer and bubbled with 98% O2-2% CO2, pH 7.8 at 20 degrees C. 3. The intracellular concentrations of the phosphorus metabolites were measured in total darkness or during prolonged exposure to light. The concentration of nucleoside triphosphates (NTP) in the dark-adapted retina was about 1.5 mM and that of phosphocreatine (PCr) was about 0.7 mM. 4. In saturating levels of light, 6.0 x 10(11) or 1.5 x 10(13) quanta s-1 cm-2 at 520 nm, the levels of PCr and phosphomonoesters rose, the levels of NTP and protons (pH) were maintained, and the levels of pyridine nucleotides and nucleotide diphosphates (NDP) fell. 5. A rise in the level of PCr in the presence of an unchanged level of NTP in the light-adapted retina indicates that the energy consumption of the retina is greater in the dark. 6. These results are in agreement with the results of oxygen consumption, glucose dependence, and electrophysiological studies which also indicate that the metabolic energy requirement of the retina decreases in light.

Adaptation, Physiological↗

PubMed1992

Cone visual pigments are present in gecko rod cells.

The Tokay gecko (Gekko gekko), a nocturnal lizard, has two kinds of visual pigments, P467 and P521. In spite of the pure-rod morphology of the photoreceptor cells, the biochemical properties of P521 and P467 resemble those of iodopsin (the chicken red-sensitive cone visual pigment) and rhodopsin, respectively. We have found that the amino acid sequence of P521 deduced from the cDNA was very similar to that of iodopsin. In addition, P467 has the highest homology with the chicken green-sensitive cone visual pigment, although it also has a relatively high homology with rhodopsins. These results give additional strength to the transmutation theory of Walls [Walls, G. L. (1934) Am. J. Ophthalmol. 17, 892-915], who proposed that the rod-shaped photoreceptor cells of lizards have been derived from ancestral cone-like photoreceptors. Apparently amino acid sequences of visual pigments are less changeable than the morphology of the photoreceptor cells in the course of evolution.

Amino Acid Sequence↗