Fast light-evoked potential from leaves.

When a leaf is illuminated with an intense flash of light, an elec trical response with a time course in milliseconds can be recorded. This re sponse was obtained between two wick electrodes placed at different positions on top of the leaf, with the entire leaf uniformly illuminated by the flash. During the first millisecond or so, the electrode nearer the apex of the leaf always became negative with respect to an electrode at the base, which indi cates that the voltage-generating source is fixed longitudinally in the leaf.

Phycomyces: stimulus storage in light-initiated reactions.

The sporangiophores of Phycomyces blakesleeanus, a unicellular fungus, increase their growth rate temporarily when given a symmetric light pulse. When a sporangiophore is cooled until normal growth stops, the light pulse can still be perceived, and, after the sporangiophore is warmed to room temperature, the normal light-growth response is observed. Thus stimulus information can be "stored" at low temperatures in this sensory system.

Isolation of an inhibitory protein for the cyclic guanosine 3','5'-monophosphate phosphodiesterase of bovine rod outer segments.

Cyclic guanosine 3',5'-monophosphate phosphodiesterase in crude extracts from bovine rod outer segments can be activated by the addition of bleached rod outer segment membranes and GTP. In the absence of rhodopsin-containing membranes, the phosphodiesterase specific activity decreases with increasing concentration. A trypsin-sensitive inhibitor believed to be responsible for this phenomenon can be separated from the phosphodiesterase by DEAE-cellulose chromatography of the crude extract. Phosphodiesterase eluted from the DEAE-cellulose column shows considerably less concentration-dependence than in the crude extract. This partially purified phosphodiesterase was used as the substrate to assay for inhibitor. A GTPase which is active only in the presence of bleached rod outer segment membranes coelutes with the phosphodiesterase and is distinct from the phosphodiesterase inhibitor we have isolated.

Resonance Raman study of the dark-adapted form of the purple membrane protein.

The resonance Raman spectrum of the dark-adapted form of the purple membrane protein (bacteriorhodopsin) has been obtained and is compared to the light-adapted pigment and model chromophore spectra. As in the light-adapted form, the chromophore-protein linkage is found to be a protonated Schiff base. Electron delocalization appears to play the dominant role in color regulation. The dark-adapted spectrum indicates a conformation closer to 13-cis than the light-adapted spectrum.

Light-initiated changes of cyclic guanosine monophosphate levels in the frog retina measured with quick-freezing techniques.

Although there is good agreement that light reduces the amount of cyclic GMP (cGMP) in the retina, the exact time-course of this decrease is not well established. Bullfrog retinal sections were isolated under infrared light and quick-frozen with liquid nitrogen-cooled, metal hammers after exposure to various intensities of continuous illumination. This quick-freezing should stop the degradation of cGMP within 50-100 ms. The frozen retinal sections were then slowly warmed up in the presence of perchloric acid to denature enzymes involved in cGmp metabolism. cGMP was determined by radioimmunoassay and comparison was made between light- and dark-adapted retinal sections from the same animal. The average cGMP concentration was 44.3 +/- 0.7 pmol cGMP/mg protein or 170.9 +/- 3.2 pmol cGMP/retina. After 1 s of illumination no significant change in cGMP concentration was found even with the brightest light used (approximately 7 x 10(7) rhodopsins bleached/second per rod. At this intensity the first significant decrease in cGMP from dark-adapted levels was detected 3-5 s after the initiation of illumination; cGMP decayed to 70-75% of the dark-adapted value after approximately 30 s. With lower intensity illumination the cGMP levels recovered to dark-adapted levels after the initial decrease even though the bleaching light remained on.

Two groups control light-induced Schiff base deprotonation and the proton affinity of Asp85 in the Arg82 his mutant of bacteriorhodopsin.

Arg(82) is one of the four buried charged residues in the retinal binding pocket of bacteriorhodopsin (bR). Previous studies show that Arg(82) controls the pK(a)s of Asp(85) and the proton release group and is essential for fast light-induced proton release. To further investigate the role of Arg(82) in light-induced proton pumping, we replaced Arg(82) with histidine and studied the resulting pigment and its photochemical properties. The main pK(a) of the purple-to-blue transition (pK(a) of Asp(85)) is unusually low in R82H: 1.0 versus 2.6 in wild type (WT). At pH 3, the pigment is purple and shows light and dark adaptation, but almost no light-induced Schiff base deprotonation (formation of the M intermediate) is observed. As the pH is increased from 3 to 7 the M yield increases with pK(a) 4.5 to a value approximately 40% of that in the WT. A transition with a similar pK(a) is observed in the pH dependence of the rate constant of dark adaptation, k(da). These data can be explained, assuming that some group deprotonates with pK(a) 4.5, causing an increase in the pK(a) of Asp(85) and thus affecting k(da) and the yield of M. As the pH is increased from 7 to 10.5 there is a further 2.5-fold increase in the yield of M and a decrease in its rise time from 200 micros to 75 micros with pK(a) 9. 4. The chromophore absorption band undergoes a 4-nm red shift with a similar pK(a). We assume that at high pH, the proton release group deprotonates in the unphotolyzed pigment, causing a transformation of the pigment into a red-shifted "alkaline" form which has a faster rate of light-induced Schiff base deprotonation. The pH dependence of proton release shows that coupling between Asp(85) and the proton release group is weakened in R82H. The pK(a) of the proton release group in M is 7.2 (versus 5.8 in the WT). At pH < 7, most of the proton release occurs during O --> bR transition with tau approximately 45 ms. This transition is slowed in R82H, indicating that Arg(82) is important for the proton transfer from Asp(85) to the proton release group. A model describing the interaction of Asp(85) with two ionizable residues is proposed to describe the pH dependence of light-induced Schiff base deprotonation and proton release.

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.

pKa of the protonated Schiff base of visual pigments.

The pKa of bovine rhodopsin is greater than 15; that of the long-wave-length-sensitive gecko P521 pigment ranges from 8.4 to 10.5 depending on chloride concentration; and that of octopus, an invertebrate, is 10.5. These pKa values are much higher than are needed just to maintain the Schiff base in its protonated state in the photoreceptor cell. The high pKa of the Schiff base may be at least partially related to a low pKa of its counterion, which would lower the frequency of thermal isomerization of the chromophore and thus lower the dark noise in the photoreceptor cell. After light absorption, the high pKa of the protonated Schiff base of a vertebrate visual pigment must get lowered enough to allow it to deprotonate, a required step in vertebrate visual excitation. This deprotonation step is not required in invertebrate visual excitation.

Trapping and spectroscopic identification of the photointermediates of bacteriorhodopsin at low temperatures.

Light-driven transmembrane proton pumping by bacteriorhodopsin occurs in the photochemical cycle, which includes a number of spectroscopically identifiable intermediates. The development of methods to crystallize bacteriorhodopsin have allowed it to be studied with high-resolution X-ray diffraction, opening the possibility to advance substantially our knowledge of the structure and mechanism of this light-driven proton pump. A key step is to obtain the structures of the intermediate states formed during the photocycle of bacteriorhodopsin. One difficulty in these studies is how to trap selectively the intermediates at low temperatures and determine quantitatively their amounts in a photosteady state. In this paper we review the procedures for trapping the K, L, M and N intermediates of the bacteriorhodopsin photocycle and describe the difference absorption spectra accompanying the transformation of the all-trans-bacteriorhodopsin into each intermediate. This provides the means for quantitative analysis of the light-induced mixtures of different intermediates produced by illumination of the pigment at low temperatures.

Biosynthetic incorporation of m-fluorotyrosine into bacteriorhodopsin.

Halobacterium halobium, grown in a defined medium where tyrosine had been largely replaced with m-fluorotyrosine, biosynthetically produced purple membrane. Analysis of this membrane by high pressure liquid chromatography of phenylthiocarbamyl derivatized amino acids of membrane acid hydrolysates revealed that up to 50% of the tyrosine was present as the m-fluorotyrosine form. Yields of the purple membrane decreased as the level of incorporation increased. The experimental purple membrane showed a single 19F NMR resonance at -61.983 ppm (relative to trifluoroacetic acid). The bacteriorhodopsin (bR) in the purple membrane was normal as assayed by gel electrophoresis, isoelectric focusing, circular dichroic spectra, and UV-visible spectra. However, the fluorinated tyrosine bacteriorhodopsins at near neutral pH exhibited slightly slower rates of proton uptake and a slower M-state decay with biphasic kinetics reminiscent of alkaline solutions of bR (pH > 9). These results imply that the tyrosines in bacteriorhodopsin may play a role in the photoactivated proton translocation process of this pigment.

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.

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.

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.

Ring oxidized retinals form unusual bacteriorhodopsin analogue pigments.

Three ring oxidized retinal analogues have been isolated from the exhaustive oxidation of all-trans retinal. All-trans 4-oxoretinal and 2,3-dehydro-4-oxoretinal have similar absorption maxima to that of all-trans retinal and have been shown to be in the 6-s-cis conformation in solution. Pigments formed with bacterioopsin exhibit absorption maxima (520 nm) blue-shifted from that of bacteriorhodopsin (bR), indicating a disturbance of the external point charge by the electronegative carbonyl moiety at the 4 position. The third analogue contains a ring contracted to a cyclopentenyl-alpha,beta-dione. Unlike the majority of retinals, this analogue displays a 6-s-trans conformation in solution and has a red-shifted absorption maximum at 435 nm. The resulting bR analogue pigment (515 nm) is formed five times faster than the other oxoretinal pigments. All three oxoretinal pigments show an irreversible 20 nm blue shift upon exposure to white light. The 4-oxo and 2,3-dehydro-4-oxoretinal pigments, after irradiation, undergo a small reversible blue shift (4-8 nm) on dark adaptation. These two pigments pump protons, although with slowed photocycle kinetics, demonstrating that these structural changes (addition of the carbonyl at the C-4 and insertion of a double bond in the ring) do not block the function of the pigment. Extraction of the C-15 tritiated analogue retinals from illuminated and non-illuminated pigments of all three oxoretinals yield identical results. Therefore, any crosslinking of these oxoretinals to the protein is by linkages which are unstable to the extraction procedures.

Mutation of arginine 134 to lysine alters the pK(a)s of key groups involved in proton pumping by bacteriorhodopsin.

Arginine 134 is located near the extracellular surface of bacteriorhodopsin (bR) and may interact with one or more nearby glutamate residues. In the bR mutant R134K, light-induced Schiff-base deprotonation (formation of the M intermediate) exhibits several kinetic components and has a complex pH dependence. The kinetics and pH dependence of M formation were analyzed using the following general guidelines for interpreting M formation: (1) The fastest component of M formation reflects the redistribution of the Schiff-base proton to D85, the usual proton acceptor, in response to the change in the proton affinities of the Schiff base and D85 early in the photocycle; (2) Two additional components of M formation reflect transitions between spectroscopically similar substates of M. By applying these guidelines, supplemented by information about the pK(a)s of D85 and the proton release group from acid (purple-to-blue) and alkaline titrations of the absorption spectra of the unphotolyzed R134K pigment, we explain the pH dependence of M formation as being due to titration of the counterion, D85, and of the proton release group. We calculate, in R134K, that the pKa of D85 is 4.6 in the unphotolyzed state, while the pKa of the proton release group is 8.0 in the unphotolyzed state but drops to approximately 5.8 in the M intermediate. The same value for the pKa of the proton release group in the M intermediate is obtained when we use photocurrent measurements to monitor proton release. The altered values of these pK(a)s relative to the corresponding values in wild-type bR suggest that D85 and the proton release group are coupled more weakly in R134K than in the wild type.

Monoclonal antibodies specific to chicken iodopsin.

Monoclonal antibodies (mABs) from hybridoma cells were raised and screened with a purified cone pigment, iodopsin, from the chicken retina. Four different methods were used to test these antibodies: (1) dot-immunobinding assay; (2) enzyme-linked immunoabsorbent assay (ELISA); (3) one dimensional immunoblotting and (4) two dimensional immunoblotting. Three classes of antibody producing cell lines were identified. One class produces a mAB specific to iodopsin. The mAB from the second class crossreacts with iodopsin and probably one of the other three cone pigments. The mAB from the third class probably crossreacts with all the four cone pigments in the chicken retina. The mABs from all these classes of hybridoma cell lines were selected so that they do not crossreact with rhodopsin. Two dimensional immunoblotting indicated that iodopsin has a much higher isoelectric point than rhodopsin, as suggested from the known amino acid sequences of human rod and cone pigments.

The unique lipid composition of gecko (Gekko Gekko) photoreceptor outer segment membranes.

This study investigated the lipid and fatty acid composition of gecko photoreceptor outer segment membranes which contain the P521 cone-type pigment. The lipids of gecko photoreceptor outer segment membranes were first extracted and separated by thin layer chromatography (TLC) and then analyzed by gas chromatography (GC). Our results show that gecko photoreceptor outer segment membranes contain less phosphatidylethanolamine (PE) and more phosphatidylcholine (PC) and phosphatidylserine (PS) compared with those of bovine and frog. The content of the polyunsaturated fatty acid, docosahexaenoic acid (DHA), in PC and PS is also the highest yet reported (55 and 63%, respectively). These lipid differences may provide some insight into the specific lipid requirements of cone-type pigments.