Masatsugu Seidou

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.

On some physicochemical properties of sucrose esters and the stability they confer to membrane proteins

Abstract A homologous series of fatty acid monoesters of sucrose whose chain length varies from 8 to 22 carbon atoms has been examined with respect to some physicochemical properties. In this series, the critical micelle concentration is comparable to that of lysophospholipids having similar hydrophobic moiety. The behavior of a spin probe dispersed in micelles of sucrose esters and their surface pressure-area isotherms at the air/water interface indicate that, despite a relatively large hydrophilic head, they form increasingly compact structures as their chain length increases. Finally, when used as dispersing medium for the purified intrinsic protein rhodopsin, long-chain esters confer to the pigment a thermal stability which is close to that of its membrane-bound state. We conclude that these detergents may be among those best suited to purified intrinsic membrane protein studies.

Structural and functional differences of two forms of GTP-binding protein, Gq, in the cephalopod retina

The major GTP-binding protein (G-protein) in the rhabdomeric photoreceptor membranes of the squid (Watasenia scintillans) has been identified as a Gq-class G-protein. Anti-Gq alpha antibodies recognized a protein not only in the photoreceptor membranes but also in soluble fractions of the retina. The 42 kD protein in the soluble fractions (soluble Gq alpha) had the same molecular mass and the same reactivities to anti-Gq antibodies as those of membrane-bound Gq alpha. The G beta subunit was scarcely detected in the soluble fractions, being found mostly in the membrane fraction, indicating soluble Gq alpha exists in monomeric form. Soluble Gq alpha had no effect on the GTPase activity of the photoreceptor membranes, suggesting that it does not interact with photoactivated rhodopsin or G beta gamma. Soluble Gq alpha would be an inactive form of Gq alpha. In the retina of Octopus fangsiao, soluble Gq alpha was scarcely detected after dark adaptation, but increased during subsequent light exposure and decreased on returning to dark adaptation. These results with Octopus suggest that functional membrane-bound Gq alpha is converted to soluble Gq alpha on exposure to light. Transformation of membrane-bound Gq alpha into the soluble form by hydroxylamine suggests that the difference between membrane-bound and soluble Gq alpha is associated with the attachment of fatty acid(s).

Luciferase activity of the intracellular microcrystal of the firefly squid, Watasenia scintillans

The arm light organ of the firefly squid, Watasenia scintillans, emits extremely bright flashes of light, which are caused by a luciferin–luciferase reaction involving ATP, Mg2+ and molecular oxygen. The molecular mechanism underlying the bioluminescence reaction has remained unresolved, because the luciferase could not be identified or isolated. The arm light organ contains numerous rod‐like bodies that are 2–6 μm long and 1–2 μm thick. This paper addresses the characterization of the extracted rod‐like body. We found that the rod‐like bodies emit the light in vitro by the luciferin–luciferase reaction. Furthermore, by using the X‐ray powder diffraction method, we confirmed that the rod‐like bodies are well‐ordered microcrystals.

Amino acid sequence of the retinal binding site of squid visual pigment

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.

Light-induced, GTP-binding protein mediated membrane currents of Xenopus oocytes injected with rhodopsin of cephalopods

Xenopus oocytes that were injected with rhabdomeric membranes of squid and octopus photoreceptors acquired light sensitivity. The injected oocytes showed a light-induced current having characteristics similar to other G-protein-mediated Cl- currents induced by the activation of other membrane receptors. Pretreatment of the oocytes with pertussis toxin before the injection suppressed the generation of the light-induced current, indicating an ability of cephalopod rhodopsin to cross-react with an endogenous G-protein of Xenopus oocytes.

Conformation changes of cuttlefish (Euprymna morsei) rhodopsin following photoconversion

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.