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Y Fukada

Publications and source records attributed to Y Fukada.

At least 127 records · Page 7Linked to original sources
PubMed1992

Photoreceptor cell types in the retina of various vertebrate species: immunocytochemistry with antibodies against rhodopsin and iodopsin.

Types of photoreceptor cells in the retinas of 36 species of vertebrates (5 classes, 14 orders) were investigated immunocytochemically with monoclonal antibodies against chicken iodopsin (Io-mAb) and antiserum against bovine rhodopsin (Rh-As). In mammals, Rh-As labeled the outer segments of some photoreceptor cells in striped squirrels (a diurnal mammal) and those of most photoreceptor cells in mice (a nocturnal mammal), while Io-mAb labeled any photoreceptor cells in either of them. In all species of birds studied, Io-mAb labeled the principal and accessory members of double cones and single cones with a red oil droplet. Rh-As labeled single cones with a yellow or clear oil droplet in addition to rods. In turtles, both Rh-As and Io-mAb labeled single cones with a red or clear oil droplet and the principal (with a yellow oil droplet) and accessory members of double cones. This suggests that the visual pigments in these cones of turtles have common epitopes with bovine rhodopsin and chicken iodopsin. In Japanese grass lizards, single cones with a yellow oil droplet and double cones were immunoreactive to both Rh-As and Io-mAb. In snakes, rods and cones could not be distinguished but both positively and negatively stained cells were observed by the use of each antibody. In geckos, however, all photoreceptor cells were immunonegative to Io-mAb. In all species studied in amphibians, Rh-As labeled rods but not cones. Neither rods nor cones reacted with Io-mAb. In fishes, almost all species studied had well developed cones, and some of these cones were labeled by Rh-As. However, Io-mAb labeled the outer segments of some cones only in loaches. Rh-As labeled photoreceptor cells in all species of fishes studied. Thus, Rh-As recognized the outer segments of rods in all species studied from fishes to mammals, whereas the epitope recognized by Io-mAb is conserved in some species of fishes, most species of reptiles and all species of birds studied.

Animals↗
PubMed1991

Carboxyl methylation and farnesylation of transducin gamma-subunit synergistically enhance its coupling with metarhodopsin II.

A heterotrimeric G-protein in vertebrate photoreceptor cells is called transducin (T alpha beta gamma), whose gamma-subunit is a mixture of two components, T gamma-1 and T gamma-2. T gamma-2 is S-farnesylated and partly carboxyl methylated at the C-terminal cysteine residue, whereas T gamma-1 lacks the modified cysteine residue. To elucidate the physiological significance of the double modifications in T gamma, we established a simple chromatographic procedure to isolate T gamma-1, methylated T gamma-2 and non-methylated T gamma-2 on a reversed phase column. Taking advantage of the high and reproducible yield of T gamma from the column, we analyzed the composition of T gamma subspecies in the T alpha-T beta gamma complex which did not bind with transducin-depleted rod outer segment membranes containing metarhodopsin II. The binding of T alpha-T beta gamma with the membranes was shown to require the S-farnesylated cysteine residue of T gamma, whose methylation further enhanced the binding. This synergistic effect was not evident when T alpha was either absent or converted to the GTP-bound form which is known to dissociate from T beta gamma. Thus we concluded that a formation of the ternary complex, T alpha-T beta gamma-metarhodopsin II, is enhanced by the farnesylation and methylation of T gamma. This suggests that the double modifications provide most efficient signal transduction in photoreceptor cells.

Amino Acid Sequence↗
PubMed1991

Effect of chitosan feeding on intestinal bile acid metabolism in rats.

The effect of chitosan feeding (for 21 days) on intestinal bile acids was studied in male rats. Serum cholesterol levels in rats fed a commercial diet low in cholesterol were decreased by chitosan supplementation. Chitosan inhibited the transformation of cholesterol to coprostanol without causing a qualitative change in fecal excretion of these neutral sterols. Increased fiber consumption did not increase fecal excretion of bile acids, but caused a marked change in fecal bile acid composition. Lithocholic acid increased significantly, deoxycholic acid increased to a lesser extent, whereas hyodeoxycholic acid and the 6 beta-isomer and 5-epimeric 3 alpha-hydroxy-6-keto-cholanoic acid(s) decreased. The pH in the cecum and colon became elevated by chitosan feeding which affected the conversion of primary bile acids to secondary bile acids in the large intestine. In the cecum, chitosan feeding increased the concentration of alpha-,beta-, and omega-muricholic acids, and lithocholic acid. However, the levels of hyodeoxycholic acid and its 6 beta-isomer, of monohydroxy-monoketo-cholanoic acids, and of 3 alpha, 6 xi, 7 xi-trihydroxy-cholanoic acid decreased. The data suggest that chitosan feeding affects the metabolism of intestinal bile acids in rats.

Animals↗
PubMed1991

Structure and function of gamma-subunit of photoreceptor G-protein (transducin).

1. The gamma-subunit of the vertebrate photoreceptor GTP-binding protein (transducin) is S-farnesylated at the C-terminal cysteine residue, with a part of the residue being methyl-esterified at the alpha-carboxyl group. 2. Functionally, the modified cysteine residue is implicated in efficient coupling of the alpha- and beta gamma-subunits, and indispensible for expressing GTP-binding activity. 3. Similar modifications, isoprenylation and methyl-esterification of the C-terminal cysteine residue have been found in a variety of proteins involved in signal transduction and growth regulation processes. However, it seems likely that the physiological roles of the modifications are different for the various proteins.

Amino Acid Sequence↗
PubMed1991

Coding visual images of objects in the inferotemporal cortex of the macaque monkey.

1. The inferotemporal cortex (IT) has been thought to play an essential and specific role in visual object discrimination and recognition, because a lesion of IT in the monkey results in a specific deficit in learning tasks that require these visual functions. To understand the cellular basis of the object discrimination and recognition processes in IT, we determined the optimal stimulus of individual IT cells in anesthetized, immobilized monkeys. 2. In the posterior one-third or one-fourth of IT, most cells could be activated maximally by bars or disks just by adjusting the size, orientation, or color of the stimulus. 3. In the remaining anterior two-thirds or three-quarters of IT, most cells required more complex features for their maximal activation. 4. The critical feature for the activation of individual anterior IT cells varied from cell to cell: a complex shape in some cells and a combination of texture or color with contour-shape in other cells. 5. Cells that showed different types of complexity for the critical feature were intermingled throughout anterior IT, whereas cells recorded in single penetrations showed critical features that were related in some respects. 6. Generally speaking, the critical features of anterior IT cells were moderately complex and can be thought of as partial features common to images of several different natural objects. The selectivity to the optimal stimulus was rather sharp, although not absolute. We thus propose that, in anterior IT, images of objects are coded by combinations of active cells, each of which represents the presence of a particular partial feature in the image.

Animals↗
PubMed1990

Phosphorylation of iodopsin, chicken red-sensitive cone visual pigment.

The amino acid sequence has been determined for the carboxyl-terminal 41 amino acids of chicken red-sensitive cone pigment, iodopsin. This sequence is distinct from but structurally homologous to that of other visual pigments. It contains a region rich in the hydroxy amino acids serine and threonine. In the related rod cell visual pigment, rhodopsin, such serines and threonines have previously been identified as sites for phosphorylation by rhodopsin kinase. Phosphorylation of photolyzed rhodopsin serves to terminate its ability to function in visual transduction as an activator of G-protein. We have purified and reconstituted both chicken rhodopsin and chicken iodopsin and shown them to be phosphorylated by bovine rhodopsin kinase. Chicken iodopsin has a Km and Vmax similar to but distinguishably different from that for bovine rhodopsin. These results, in conjunction with other data, suggest that visual pigments in cone cells, upon absorption of light, undergo functional processes similar to those of the visual pigments in rod cells.

Amino Acid Sequence↗
PubMed1990

The primary structure of iodopsin, a chicken red-sensitive cone pigment.

A purified iodopsin was digested by CNBr or several proteolytic enzymes into fragments, the amino acid sequences of which were determined. A partial sequence of the C-terminal fragment was utilized for synthesizing an oligonucleotide probe which identified the iodopsin cDNA (1339 bases). The deduced amino acid sequence (362 residues) had 80%, 42%, or 43% homology to that of human red-sensitive cone pigment, cattle or chicken rhodospin, respectively. Although the hydropathy profile implies that iodopsin, like rhodopsin, has 7 transmembrane alpha-helical segments, iodopsin may have a hydrophilic pocket near the seventh segment on the basis of the unexpected cleavages in the middle of the segment VII by chymotrypsin under nondenaturing conditions.

Amino Acid Sequence↗
PubMed1990

Structure of the retinal chromophore in 7,9-dicis-rhodopsin.

Bovine rhodopsin was bleached and regenerated with 7,9-dicis-retinal to form 7,9-dicis-rhodopsin, which was purified on a concanavalin A affinity column. The absorption maximum of the 7,9-dicis pigment is 453 nm, giving an opsin shift of 1600 cm-1 compared to 2500 cm-1 for 11-cis-rhodopsin and 2400 cm-1 for 9-cis-rhodopsin. Rapid-flow resonance Raman spectra have been obtained of 7,9-dicis-rhodopsin in H2O and D2O at room temperature. The shift of the 1654-cm-1 C = N stretch to 1627 cm-1 in D2O demonstrates that the Schiff base nitrogen is protonated. The absence of any shift in the 1201-cm-1 mode, which is assigned as the C14-C15 stretch, or of any other C-C stretching modes in D2O indicates that the Schiff base C = N configuration is trans (anti). Assuming that the cyclohexenyl ring binds with the same orientation in 7,9-dicis-, 9-cis-, and 11-cis-rhodopsins, the presence of two cis bonds requires that the N-H bond of the 7,9-dicis chromophore points in the opposite direction from that in the 9-cis or 11-cis pigment. However, the Schiff base C = NH+ stretching frequency and its D2O shift in 7,9-dicis-rhodopsin are very similar to those in 11-cis- and 9-cis-rhodopsin, indicating that the Schiff base electrostatic/hydrogen-bonding environments are effectively the same. The C = N trans (anti) Schiff base geometry of 7,9-dicis-rhodopsin and the insensitivity of its Schiff base vibrational properties to orientation are rationalized by examining the binding site specificity with molecular modeling.

Animals↗
PubMed1990

Farnesylated gamma-subunit of photoreceptor G protein indispensable for GTP-binding.

Transducin, composed of subunits T alpha, T beta and T gamma, is a member of a heterotrimeric G-protein family, and transduces the light signal in visual cells. We have recently found that bovine T beta gamma can be separated into two components. T beta gamma-1 and T beta gamma-2, each of which has its own gamma-subunit, T gamma-1 and T gamma-2, respectively. T beta gamma-2 enhances the binding of GTP to T alpha in the presence of metarhodopsin II by about 30-fold compared with T beta gamma-1. Here we show that a farnesyl moiety is attached to a sulphur atom of the C-terminal cysteine of T gamma-2 (active form), a part of which is additionally methyl-esterified at the alpha-carboxyl group. In T gamma-1 (inactive form), however, such modifications are missing. Thus, the farnesyl moiety attached to the gamma-subunit is indispensable for the GTP-binding activity of transducin. This suggests that a similar modification may occur in the gamma-subunits of other heterotrimeric G proteins involved in biological signal transduction processes.

Amino Acid Sequence↗
PubMed1990

Effects of chloride on chicken iodopsin and the chromophore transfer reactions from iodopsin to scotopsin and B-photopsin.

Spectroscopic properties of chicken iodopsin were investigated in correlation with the concentration of chloride in digitonin extracts. When chloride in the extract was depleted by extensive dialysis, chloride-depleted iodopsin (absorption maximum, 512 nm) was formed. It was converted to chloride-bound iodopsin (absorption maximum, 562 nm) by the addition of chloride in the extract. There existed an equilibrium between two forms of iodopsin with a dissociation constant of 0.8 mM chloride. The chromophore-transfer reaction from iodopsin to scotopsin or B-photopsin, the protein moiety of chicken rhodopsin or chicken blue-sensitive cone pigment, respectively, in digitonin extract was also investigated in correlation with the concentrations of chloride, other monovalent and divalent anions, and detergent. The chromophore of chloride-depleted iodopsin was easily transferred to scotopsin in the extract, resulting in formation of rhodopsin. On the other hand, chloride-bound iodopsin was fairly stable even in the presence of scotopsin, indicating that the reaction is inhibited by binding of chloride to iodopsin. The chromophore-transfer reaction to B-photopsin was also observed from chloride-depleted iodopsin but not from chloride-bound iodopsin. The reaction was observable in the 10% digitonin extract as well as in the 2% digitonin extract. The reaction was also observed when 25 mM Na2SO4 was present in the mixture instead of NaCl, but was not when 67 mM NaNO3 was present. All these facts suggest that the chloride binding site of iodopsin does not accept a divalent anion such as SO4(2+), but does accept a monovalent anion such as Cl- or NO3-, which causes inhibition of the chromophore transfer.

Absorption↗
PubMed1990

A specific beta gamma-subunit of transducin stimulates ADP-ribosylation of the alpha-subunit by pertussis toxin.

Transducin (T alpha and T beta gamma) is a GTP-binding protein involved in the visual transduction process in a rod outer segment. We have previously demonstrated that T beta gamma is a mixture composed of two components, T beta gamma-1 and T beta gamma-2, with distinctive gamma-subunits, T gamma-1 and T gamma-2, respectively (Fukada et al., 1989, J. Biol. Chem., 264, 5937-5943). To investigate the interaction between T alpha and the two components of T beta gamma, the effect of either T beta gamma-1 or T beta gamma-2 on the ADP-ribosylation of T alpha catalyzed by pertussis toxin (IAP) was examined. T beta gamma-2 stimulated the ADP-ribosylation of T alpha by IAP, while T beta gamma-1 displayed almost no enhancement of the ADP-ribosylation. Addition of T beta gamma-1 to the mixture of T alpha and T beta gamma-2 had no effect on the ADP-ribosylation of T alpha. These results indicate that T alpha and T beta gamma-2 form a complex that serves as a substrate of IAP in the ADP-ribosylation reaction, while T beta gamma-1 has a little affinity for T alpha. It was suggested that T gamma-2 is an essential subunit for T beta to interact with T alpha.

Adenosine Diphosphate Ribose↗
PubMed1990

Comparative study on the chromophore binding sites of rod and red-sensitive cone visual pigments by use of synthetic retinal isomers and analogues.

A comparative study on the chromophore (retinal) binding sites of the opsin (R-photopsin) from chicken red-sensitive cone visual pigment (iodopsin) and that scotopsin) from bovine rod pigment (rhodopsin) was made by the aid of geometric isomers of retinal (all-trans, 13-cis, 11-cis, 9-cis, and 7-cis) and retinal analogues including fluorinated (14-F, 12-F, 10-F, and 8-F) and methylated (12-methyl) 11-cis-retinals. The stereoselectivity of R-photopsin for the retinal isomers and analogues was almost identical with that of scotopsin, indicating that the shapes of the chromophore binding sites of both opsins are similar, although the former appears to be somewhat more restricted than the latter. The rates of pigment formation from R-photopsin were considerably greater than those from scotopsin. In addition, all the iodopsin isomers and analogues were more susceptible to hydroxylamine than were the rhodopsin ones. These observations suggest that the retinal binding site of iodopsin is located near the protein surface. On the basis of the spectral properties of fluorinated analogues, a polar group in the chromophore binding site of iodopsin as well as rhodopsin was estimated to be located near the hydrogen atom at the C10 position of the retinylidene chromophore. A large difference in wavelength between the absorption maxima of iodopsin and rhodopsin was significantly reduced in the 9-cis and 7-cis pigments. On the assumption that the retinylidene chromophore is anchored rigidly at the alpha-carbon of the lysine residue and loosely at the cyclohexenyl ring, each of the two isomers would have the Schiff-base nitrogen at a position altered from that of the 11-cis pigments.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Binding of GTP to transducin is not inhibited by arrestin and phosphorylated rhodopsin.

In the presence of a photobleaching intermediate of unphosphorylated or phosphorylated rhodopsin (Rh*), the binding of GppNHp to transducin was measured with or without arrestin for elucidation of the shut-off mechanism of the visual transduction process in bovine rod outer segments. The ability of Rh* to catalyze the formation of the transducin-GppNHp complex in the absence of arrestin was independent of the degree of phosphorylation of Rh*. Furthermore, the catalyzing ability of the phosphorylated Rh* was not reduced by the addition of arrestin. These observations indicate that the interaction between phosphorylated Rh* and transducin was not inhibited by arrestin. Thus, the hypothesis was not supported that the PDE shut-off process is a simple competition between transducin and arrestin for binding to phosphorylated Rh*.

Animals↗
PubMed1990

Functional heterogeneity of beta gamma-subunit of frog transducin.

1. Transducin subunits (T alpha and T beta gamma) were purified from freshly dissected frog (Rana catesbeiana) retinas. It was found that purified T beta gamma is composed of three components which can be separated from each other by an anion exchange column chromatography under nondenaturing conditions. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of these three components demonstrated that each contains T beta (mol. wt 35,000) and T gamma (mol. wt approximately 8000). 3. Only one of the three components retained an ability to enhance the binding of GppNHp to T alpha in the presence of a photobleaching intermediate of rhodopsin, while the others showed very low abilities to enhance the binding. 4. These observations, together with the similar findings on bovine T beta gamma, strongly suggest that the functional heterogeneity of T beta gamma is conserved in vertebrate photoreceptor cells.

Animals↗
PubMed1990

Bathoiodopsin, a primary intermediate of iodopsin at physiological temperature.

Measurement of the primary photochemical reaction of iodopsin, a chicken red-sensitive cone visual pigment, was carried out at room temperature by using picosecond (ps) laser photolysis. Excitation of iodopsin with a ps green pulse (pulse width, 21 ps) caused the instantaneous formation of a bathochromic product, which was stable on a ps time scale. This product may correspond to "bathoiodopsin," which was detected by low-temperature spectrophotometry. Although bathoiodopsin produced at the temperature of liquid nitrogen or helium reverted to the original pigment (iodopsin) on warming (above -170 degrees C), the bathoiodopsin produced at physiological temperature decayed to all-trans-retinal and R-photopsin (the protein moiety of iodopsin) presumably through several intermediates. The absorption maximum of bathoiodopsin at room temperature was at 625 nm, a wave-length slightly shorter than that measured at low temperature (lambda max, 640 nm). The extinction coefficient of bathoiodopsin at room temperature was lower than that at low temperature and close to that of the original iodopsin at room temperature.

Animals↗
PubMed1990

Localization of iodopsin in the chick retina during in vivo and in vitro cone differentiation.

Using highly specific antibodies against a chick red-sensitive cone pigment, iodopsin, we investigated the localization of iodopsin in the developing and mature chick retina. The chick retina contains several different photoreceptor types, including a rod, a double cone with a principal and accessory cone, and four different types of single cones. Immunocytochemical observations revealed that outer segments (OS) of one of the single cones (type 1) and both cells of the double cone were strongly immunoreactive to anti-iodopsin antibodies. The Golgi regions and small vesicular structures in the inner segments (IS) of these cells also were intensely stained, indicating a continuous synthesis of iodopsin and its addition to the newly formed cone OS. In the differentiating cones of the developing but immature chick retina, iodopsin immunoreactivity was found at the plasma membranes of both the IS and the terminals (pedicles). This suggests that unidirectional transport of iodopsin to the outer segment may be established during cone differentiation. Immunostaining in the outer plexiform layer (OPL) produced two bands, suggesting that the pedicles of the double cones and type 1 single cones terminate at different positions in this layer. Application of the antibodies to a cell culture system of the chick retina revealed that cells immunoreactive to anti-iodopsin differ slightly in morphology from those reactive with anti-rhodopsin. Since antibodies to iodopsin and rhodopsin stained different types of photoreceptors in the intact chick retina, it will be possible to analyze cell lineage of rods and cones in vitro by use of these antibodies.

Animals↗