Yuji Kito

Phase transitions of the purple membrane and the brown holo-membrane X-ray diffraction, circular dichroism spectrum and absorption spectrum studies

Abstract The phase transition of the purple membrane observed by differential scanning calorimetry (Jackson, M.B. and Sturtevant, J.M. (1978) Biochemistry 17, 911–915) has been investigated by X-ray diffraction, circular dichroism and absorption spectrum, in comparison with the phase transition in the brown holo-membrane. The two-dimensional crystal of bacteriorhodopsin transformed into two-dimensional liquid around 74–78°C in the purple membrane and around 50–60°C in the brown holo-membrane. The X-ray diffraction patterns obtained at 78°C for the purple membrane and at 60°C for the brown holo-membrane exhibit several broad peaks. Analysis of the pattern suggests that bacteriorhodopsin molecules aggregate in trimers even above the phase transition temperature. The negative circular dichroism band in the visible region is still present at 80°C in the purple membrane and at 60°C in the brown holo-membrane, but becomes negligibly small at 70°C in the brown holo-membrane. The 560 nm absorption peak due to bacteriorhodopsin changes its position and height drastically around 80°C in the brown holo-membrane as in the purple membrane. X-ray diffraction studies have been made on membranes of total lipids extracted from the purple membrane. No indication of the phase transition has been found between −81°C and 77°C.

On the three visual pigments in the retina of the firefly squid, Watasenia scintillans

SummaryThe deep-sea bioluminescent squid, Watasenia scintillans, has three visual pigments: The major one (A1 pigment) is based on retinal and has λmax = 484 nm, the second one (A2 pigment) is based on 3-dehydroretinal and has λmax = 500 nm, and the third one (A4 pigment) is based on 4-hydroxyretinal and has λmax = 470 nm. The distribution of these 3 visual pigments in the retina was studied by HPLC analysis of the retinals in retina slices obtained by microdissection. It was found that A1 pigment was not located in the specific region of the ventral retina receiving the down-welling light which contains very long photoreceptor cells, forming two strata. A2 and A4 pigment were found exclusively in the proximal pinkish stratum and in the distal yellowish stratum. The role of these pigments in the retina is hypothesized to involve spectral discrimination. The extraction and analysis of retinoids to determine the origin of 3-dehydroretinal and 4-hydroxyretinal in the mature squid showed only a trace amount of 4-hydroxyretinol in the eggs. Similar analysis of other cephalopods collected near Japan showed the absence of A2 or A4 pigment in their eyes.

4-Hydroxyretinal, a new visual pigment chromophore found in the bioluminescent squid, Watasenia scintillans

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.

Studies on cephalopod rhodopsin. Fatty acid esters of sucrose as effective detergents.

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.

Circular dichroism of squid rhodopsin

Abstract The linkage between 11- cis retinal and opsin in squid rhodopsin was studied by means of the measurement of circular dichroism (CD). The CD revealed by 11- cis retinal when bound with opsin is probably derived from the stereospecific interaction between them. The magnitude and the shape of the CD spectrum of squid rhodopsin in the visible range are different from those of cattle rhodopsin. This fact may be due to the difference in protein conformation, especially around the attachment site of retinal, and this is probably related to the stability of the linkage of retinal and opsin in those visual pigments. On the other hand, squid rhodopsin shows a complicated negative Cotton effect in the ultraviolet range, as is generally found in other proteins or polypeptides containing an α-helix. On illumination, squid rhodopsin loses its optical activity in the visible range at the metarhodopsin stage. The ultraviolet CD spectrum does not change, though the absorbance near 233 nm is diminished. This is ascribed to a small conformational change in the protein part. From the above results it is concluded that the conformation of 11- cis retinal is influenced by the asymmetric conformation of the protein near the retinal and the optical activity is induced. Retinal becomes optically inactive at the metarhodopsin stage because the spatial arrangement between retinal and opsin has been changed by photoisomerization.

Molecular weight and structural studies on cephalopod rhodopsin

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.

Phosphatidyl inositol-phospholipase C in squid photoreceptor membrane is activated by stable metarhodopsin via GTP-binding protein, Gq

Phosphatidyl inositol-phospholipase C (PI-PLC) in squid retina was studied by immunoblotting and its activities were determined using [3H]phosphatidyl inositol bisphosphate ([3H]PIP2) as substrate. PI-PLC activity was found mostly in soluble fraction when the retina homogenate was treated with 400 mM KCl, but was associated with rhabdomal membranes under low salt conditions (20 mM Hepes). A protein with apparent molecular mass of 130kD was recognized by an antibody against PLC beta 4/norp A in both 400 mM KCl soluble and rhabdomal membrane fractions. A 42 kD protein recognized by antibody against the C-terminus of Gq alpha was also present in these two fractions. GTP gamma S stimulated only the PI-PLC activity associated with membrane and was magnesium dependent. PI-PLC activity was found to be (i) highly dependent upon calcium concentrations, (ii) enhanced by GTP but not by other nucleotides, and (iii) significantly stimulated by light at lower concentrations of GTP gamma S. The stimulation by light was still observed when irradiated membrane was incubated at 10 degrees C for 10 min and then mixed with GTP gamma S. These results suggest that stable metarhodopsin stimulates a PLC beta 4/norp A-like enzyme via a G-protein, Gq.

On the labile intermediate of rhodopsin as demonstrated by low temperature illumination.

Abstract After illumination and re-illumination with interference filters at —78° and —195°, the absorption spectrum of rhodopsin solutions was determined at 23° in the region between 460 mμ and 520 mμ. The trend was that, after exposure of the samples to lights of different wavelengths, the shorter the wavelength was, the more the absorptions decreased. Illumination with longer wavelengths (> 550 mμ ) at —195° resulted only in a slight decrease in absorption, while illumination at —78° caused much more decrease in absorption (about 60% in relative absorbancy at 500 mμ). When a rhodopsin solution, which had been illuminated with light of a shorter wavelength, was re-illuminated with light of a longer wavelength, its absorption was increased to the level of the solution illuminated only once with the light of a longer wavelength. It was concluded from these facts that the solution illuminated at low temperature was a photochemical steady-state mixture composed of rhodopsin, iso-rhodopsin and the labile fraction, which is thermolabile at low temperature. The yield of these three components might be determined by the wavelength of light as well as temperature, with both affect the specific absorption of the labile fraction. The production of iso-rhodopsin in the illuminated solution was also examined.

Volatile anesthetics cause conformational changes of bacteriorhodopsin in purple membrane

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.

Circular dichroism of visual pigment analogues containing 3-dehydroretinal and 5,6-epoxy-3-dehydroretinal as the chromophore

Two isomers isolated from irradiated all-trans-3-dehydroretinal were coupled with opsin to form visual pigment analogues. These pigments showed λmax at 517 nm (3-dehydrorhodopsin) and 500 nm (3-dehydroisorhodopsin), respectively. In the case of 5,6-epoxy-3-dehydroretinal also, two isomers were isolated and coupled with opsin to form 5,6-epoxy-3-dehydrorhodopsin and 5,6-epoxy-3-dehydroisorhodopsin. These pigments showed λmax at almost the same wavelength of 465 nm. These synthetic pigments exhibited induced circular dichroism (CD) at the range 300–600 nm like rhodopsin and isorhodopsin. The magnitude of CD or the rotational strength on the α band of 5,6-epoxy-3-dehydrorhodopsin is about equal to that of rhodopsin, but that of 3-dehydrorhodopsin is larger. From the above results and those reported elsewhere, it is proposed that the optical activity of the chromophore in the visual pigment or its analogue is not induced by the preferential selection of an inherently twisted form of retinal.