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We quantitatively assessed the spectroscopic changes of purple membrane in relation to the concentrations of a volatile anesthetic. As reported previously, volatile anesthetics show three modes of action on purple membrane. By using an anesthetic for which the concentration in solution could be determined spectroscopically and by applying modified analytical methods regarding the M-intermediate lifetime, we were able to clarify the quantitative relation between anesthetic concentration and each mode of action, a relation which in the past has only been described qualitatively. We also determined through the measurement of transient pH changes with pyranine that the proton pump efficiency per photochemical cycle in an action mode induced with low concentrations of anesthetic does not change from that of the native state. Moreover, we dynamically obtained the individual M-bacteriorhodopsin difference spectrum of each state at room temperature using our flash photolysis system equipped with a wavelength-tunable dye laser. These results demonstrated again that we should clearly distinguish different action modes of anesthetics according to their concentrations.

Cuttlefish (Euprymna morsei) rhodopsin solubilized in lauryl ester of sucrose and its photoproduct, acid metarhodopsin, were examined by small-angle X-ray scattering and chromatofocusing to investigate the conformation changes of visual pigment following photoconversion. From spectroscopic studies, it was found that more than 93% of Euprymna rhodopsin could be converted to meta form under the condition of red light irradiation at neutral pH. Since almost pure acid metarhodopsin solution was prepared without changing the specimen concentration, the small-angle X-ray scattering intensities of both pigment-detergent complexes were directly compared. The radius of gyration increased on going from rhodopsin to acid metarhodopsin by approximately 1.5%. There were also discernible changes in the secondary peak intensities. The distribution function, derived by the Fourier transformation of intensity data, showed a significant change around 55 A. The maximum linear dimension of the rhodopsin-detergent complex was about 95 A and hardly changed after illumination. Intensity at zero angle did not change after illumination, suggesting that the aggregation did not occur. The change of the intensity profile could be due to the conformational change of the pigment-detergent monomers. The pI value of rhodopsin determined by chromatofocusing was 5.32 and that of acid metarhodopsin was 5.06, indicating that a few carboxyl groups are newly dissociated. The shift of the protein mass and the charge redistribution were observed following photoconversion.

The protein moiety of squid (Watasenia scintillans) rhodopsin has been shown to have a molecular weight of 46 800 by means of amino acid analysis. This value was comparable to the value (51 000) obtained from SDS-polyacrylamide gel electrophoresis. After the squid eyes were incubated at 10 degrees C for 8 days, the rhodopsin showed a molecular weight of 39 000 on electrophoresis. The smaller molecular weight was ascertained by amino acid analysis of the rhodopsin; and may result from autolysis by the lysosomal enzyme. The rhodopsin in rhabdomeric membranes and in detergent solution was treated with chymotrypsin, papain or subtilisin. These enzymes first produced the 39 000 dalton rhodopsin and then cleaved this into the 25 000 and 14 000 dalton peptides without bleaching. The rhodopsin was attacked by proteases and readily lost an approx. 12 000 dalton peptide portion. This portion included the COOH-terminal and was rich in glutamic acid, proline, glycine, alanine and tyrosine residues.

The retinylpeptides of visual pigments of two species of squid were identified in invertebrate visual pigments. Their primary structures were identical: H-Phe-Ala-Lys-Ala-Ser-Ala-Ile-His-Asn-pro-Hse(Met)-OH. The sequence was homologous to those of the corresponding region of other visual pigments, but the eighth amino acid, His, was found in squid visual pigments. In this experiment the retinylpeptides of eleven amino acid residues were isolated by monitoring the absorbance spectrum of the reduced retinal Schiff base without using radio-active [3H]retinal. This method is valid for the isolation and identification of retinylpeptides of other invertebrate visual pigments in which the chromophore is not exchangeable.

Photoisomerization of the chromophore of squid rhodopsin is dependent upon the irradiation temperature. Above 0 degrees C, only 11-cis in equilibrium all-trans reaction proceeds and the all-trans leads to 9-cis reaction is limited to extremely low efficiency. At liquid nitrogen temperature, 11 cis in equilibrium all-trans in equilibrium 9-cis reaction takes place. At intermediary low temperatures (-80 degrees C to -15 degrees C) another isomer of retinal may be produced by the irradiation, which forms a pigment having an absorbance maximum at 465 nm (P-465). The formation of P-465 decreases remarkably in the narrow temperature range from -30 degrees C to 0 degrees C where mesorhodopsin converts to metarhodopsin. Medsorhodopsin is quite different from metarhodopsin in the photoisomerization of the chromophore because P-465 is produced from the former but not from the latter. No P-465 is produced both at liquid nitrogen temperature and above 0 degrees C. P-465 is more labile than any of the other photoproducts so far known, that is isorhodopsin, alkaline and acid metarhodopsins. P-465 is converted to metarhodopsin by irradiation.

The ultraviolet absorbance of squid and octopus rhodopsin changes reversibly at 234 nm and near 280 nm in the interconversion of rhodopsin and metarhodopsin. The absorbance change near 280 nm is ascribed to both protein and chromophore parts. Rhodopsin is photoregenerated from metarhodopsin via an intermediate, P380, on irradiation with yellow light (lamda >520 nm). The ultraviolet absorbance decreases in the change from rhodopsin to metarhodopsin and recovers in two steps; mostly in the process from metarhodopsin to P380 and to a lesser extent in the process from P380 to rhodopsin. P380 has a circular dichroism (CD) band at 380 nm and its magnitude is the same order as that of rhodopsin. Thus it is considered that the molecular structure of P380 is close to that of rhodopsin and that the chromophore is fixed to opsin as in rhodopsin. In the change from metarhodopsin to P380, the chromophore is isomerized from the all-trans to the 11-cis form, and the conformation of opsin changes to fit 11-cis retinal. In the change from P380 to rhodopsin, a small change in the conformation of the protein part and the protonation of the Schiff base, the primary retinal-opsin link, occur.

Squid rhodopsin was extracted with solutions of fatty acid esters of sucrose (monolaurate and monostearate) and purified by DEAE-cellulose and concanavalin A-Sepharose affinity chromatography. The purified rhodopsin (A280/A480 = 2.5) contained 2.3 mol of glucosamine and 1.2 mol of phospholipid per mol of rhodopsin. The photoproduct metarhodopsin was also stable in these detergent solutions as in digitonin solution. Concanavalin A had no affinity for retinochrome.

Purified bovine rod outer segment membrane was solubilized in a mixture of 1.5% cholic acid/20% saturated ammonium sulfate and 0.05 M phosphate buffer (pH 7.5). The solubilized rod outer segment membrane was fractionated with ammonium sulfate and 70--90% of rhodopsin (A278/A498= 1.6--1.9) was recovered in the fraction of 50 to 60% saturation with ammonium sulfate, giving a highly concentrated solution of purified rhodopsin (A1CM 498 = 83). By the method of ammonium sulfate salting-out, the solubilized rod outer segment membrane was divided into several fractions without a loss of components. The components in each fraction were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Rhodopsin and opsin amounted to 93% of the total protein in the membrane. Other proteins with molecular weights of 46 000, 52 000, 56 000, 70 000, 95 000, 105 000, 130 000 and 270 000 were also detected. Most of phospholipids in the rod outer segment membrane remained in the supernatant above 60% saturation with ammonium sulfate.

The aggregation state of reconstituted bacteriorhodopsin molecules in the brown membrane has been investigated by X-ray diffraction and CD spectra. It has been confirmed that reconstituted bacteriorhodopsin molecules form the hexagonal lattice spontaneously whereas bacterioopsin molecules do not.

In the visual pigment, the chromophore retinal is bound through a Schiff base linkage with the protein moiety, opsin. The secondary interaction of retinal with opsin was studied in cephalopod rhodopsin and its photoproduct, metarhodopsin. The pK of the protonation of the Schiff base in metarhodopsin was affected by the phospholipids and detergents surrounding the protein, and varied between 9.2 and 6.9. Among nonionic detergents, the fatty acid ester of sucrose behaved like phospholipids but other detergents changed the protein conformation so that the pK of the Schiff base in metarhodopsin became nearly equal to the pK of N-retinylidene butylamine. This tendency was manifested in rhodopsin as the formation of a 380 nm pigment.

The bioluminescent squid, Watasenia scintillans has three visual pigments. The major pigment, based on retinal (lambda max 484 nm), is distributed over the whole retina. Another pigment based on 3-dehydroretinal (lambda max approximately 500 nm) and the third pigment (lambda max approximately 470 nm) are localized in the specific area of the ventral retina just receiving the downwelling light. Visual pigment was extracted and purified from the dissected retina. The chromophores were then extracted and analyzed with HPLC, NMR, infrared and mass spectroscopy, being compared with the synthetic 4-hydroxyretinal. A new retinal derivative, 11-cis-4-hydroxyretinal, is identified as the chromophore of the third visual pigment of the squid.

We examined the effects of volatile anesthetics on the structure of the bacteriorhodopsin in the purple membrane by measurements of the absorption spectrum and the visible circular dichroism (CD) spectrum and assay of the retinal composition. As the concentrations of halothane, enflurane and methoxyflurane were increased, the absorption at 560 nm decreased but that at 480 nm increased with an isosbestic point around 510 nm. These anesthetic-induced spectroscopic changes were reversible. The CD spectrum showed the biphasic pattern with a positive and a negative band. As the concentration of halothane was increased from 4 mM to 8mM, the negative band reversibly diminished more drastically than the positive band, and at 8 mM of halothane the positive band shifted to around 480 nm. These results show that halothane disturbed the exciton coupling among bacteriorhodopsin molecules. The retinal isomer composition was analyzed using high performance liquid chromatography. The ratio of 13-cis- to all-trans-retinal was 47:53, 34:66 and 19:81 at control, 7.4 mM and 14.9 mM enflurane, respectively. After elimination of enflurane, the ratio returned to the control value. These findings indicate that volatile anesthetic directly affect a bacteriorhodopsin in the purple membrane and induce conformational changes in it.