Biomedical subjects

Y Fukada

Publications and source records attributed to Y Fukada.

At least 109 records · Page 6Linked to original sources

PubMed1994

Effects of carboxyl methylation of photoreceptor G protein gamma-subunit in visual transduction.

G protein gamma-subunits are isoprenylated and carboxyl-methylated at the C-terminal cysteine, which is indispensable for the function of photoreceptor G protein transducin (T alpha beta gamma). However, the physiological role of the methylation and its reversibility have been unclear. Here we isolated methylated and non-methylated forms of farnesylated T beta gamma, and demonstrated that the methylation remarkably facilitates not only the membrane association of T beta gamma but also the subunit interaction between T alpha and T beta gamma. Consequently, the functional coupling of transducin with light-activated receptor, metarhodopsin II, was stabilized by the methylation, resulting in acceleration of GTP gamma S (guanosine 5'-3-O-(thio) triphosphate) binding to T alpha. An examination of the reversibility of the methylation suggested that T gamma is kept fully methylated in rod outer segments. These observations indicate that the methylation of T gamma plays an important role in the most efficient photon-signal transduction process in rod cells.

Adenosine Diphosphate Ribose↗

PubMed1994

Visual pigments in the pineal complex of the Japanese quail, Japanese grass lizard and bullfrog: immunocytochemistry and HPLC analysis.

We investigated localization of visual pigments in the pineal complex of Japanese quail, Japanese grass lizards and bullfrogs immunocytochemically by use of the antiserum against bovine rhodopsin (Rh-As) and monoclonal antibodies against chicken iodopsin (Io-mAb). We also analyzed retinoids, chromophores of visual pigments, by a high performance liquid chromatography (HPLC). The outer segments and cell membranes of some photoreceptor cells in the pineal organ of the Japanese quail exhibited immunoreactivity to Rh-As, but there are also many immunonegative cells. The number of immunoreactive cells among individuals varied. Immunoreactivity to Io-mAb was weak or did not exist. The HPLC analysis revealed peaks of 11-cis and all-trans isomers of retinal in the oxime extracts of the pineal organ of Japanese quail and chickens. In the pineal of Japanese grass lizards, the outer segments of some cells were immunopositive to Io-mAb, but there were no cells immunoreactive to Rh-As. The parietal eye exhibited a well-developed lens and photoreceptor cells, but the outer segments of photoreceptor cells were immunonegative to both Rh-As and Io-mAb. In bullfrogs, three types of cells were identified in both the pineal and frontal organ; (1) immunopositive to Rh-As, (2) immunopositive to Io-mAb and (3) immunonegative to either of the antibodies. In the pineal organ of bullfrogs, 11-cis and all-trans retinal and 11-cis 3-dehydroretinal were detected, and 11-cis and all-trans retinal were also detected in the frontal organ. We detected 11-cis and all-trans retinal in the ventral part of diencephalon including the hypothalamus. Thus, the chromophore is the same between the retinal and pineal visual pigments, but the expression of opsins is different between the retina and pineal complex, which probably reflects the different function of each organ.

Animals↗

PubMed1994

Mechanism of suppression of cloned human suppressor T cells.

We report the mechanism of suppression of suppressor T cell clone III-1-C5 using helper T cell clone III-1-B6, mitogen responses and rIL-2. Clone III-1-C5 suppressed the mixed lymphocyte reaction (MLR) by secreting alloantigen non-specific, MHC non-restricted suppressor factor(s). Clone III-1-C5 did not suppress mitogen (PHA, Con A, PWM) response nor proliferation by exogeneous rIL-2. Clone III-1-C5 suppressed proliferation by clone III-1-B6, which augments proliferation by direct cell to cell contact with responder cells and not by soluble factors. These results indicated that suppressor T cells exhibit suppressive effects not only by inhibiting IL-2 synthesis but by inhibiting the direct effects of helper T-cells.

Animals↗

PubMed1994

[Covalent lipid modifications of heterotrimeric G proteins].

Guanine nucleotide-binding regulatory proteins (heterotrimeric G proteins) are composed of alpha-, beta- and gamma- subunits, and they mediate a variety of intracellular signal transductions by coupling activated membrane receptors with effector enzymes and channels. Activated receptors catalyze the exchange of GDP bound to the alpha-subunits for cytosolic GTP, and GTP-bound alpha-subunits in turn regulate activities or functions of the effectors. The beta gamma-complex is not dissociable under physiological conditions, and it is indispensable for the GDP/GTP exchange reaction on the alpha-subunit. Recently, three kinds of lipid modifications have been found in the alpha- and gamma-subunits. The first is the attachment of fatty acids, myristate (C14:0) or structurally related fatty acids to the N-terminal glycine residues of some members of the alpha-subunits. Another type of fatty acylation to be characterized is the linkage of palmitate (C16:0) to a number of alpha-subunits via a thioester bond at their cysteine residues. The third type of modification is polyisoprenylation (farnesylation or geranylgeranylation) and alpha-carboxyl methylation at the C-terminal cysteine residue of the gamma-subunit. These modifications on the two subunits have been shown to play a critical role in not only protein-membrane interaction but also proper protein-protein interaction, both of which are required for the G protein function.

Amino Acid Sequence↗

PubMed1994

Immunoreactivities to rhodopsin and rod/cone transducin antisera in the retina, pineal complex and deep brain of the bullfrog, Rana catesbeiana.

Birds and lower vertebrates are known to have extra-retinal photoreceptors in the pineal complex and deep brain. Although the photoreceptive function of the pineal complex has been investigated well, the exact location and nature of the deep brain photoreceptors are not known. In this study we tried to localize visual pigments and signal transduction proteins immunohistochemically in the brain of bullfrogs (Rana catesbeiana). The retina, and the brain with the pineal and the frontal organ were fixed with Zamboni's fixative and/or Bouin's solution. Immunoreactivities to three antisera against bovine rhodopsin (Rh-As), alpha-subunits of bovine rod (anti-pTr alpha) and cone transducin (anti-pTc alpha) were shown in the retina, pineal, frontal organ and hypothalamus. The retina and pineal were immunopositive to both Rh-As and anti-pTr alpha, whereas the frontal organ was immunopositive to only Rh-As and the hypothalamus was immunopositive to all three antisera. The cells which were immunoreactive to Rh-As, anti-pTr alpha and anti-pTc alpha were observed in the preoptic nucleus and suprachiasmatic nucleus in the hypothalamus. The shape of these immunoreactive cells in the hypothalamus was round or spindle-like with one or two immunoreactive nerve processes most of which were perpendicular to the ventricular surface. Western blot analysis of the hypothalamus, pineal and frontal organ demonstrated immunoreactive bands molecular weight of which corresponded to those of the retina (34 kDa, 38 kDa and 41 kDa). Thus, visual pigments and transducin-like proteins seem to exist in the hypothalamus as well as the pineal complex of frogs.

Animals↗

PubMed1993

Nanosecond laser photolysis of iodopsin, a chicken red-sensitive cone visual pigment.

The photobleaching process of iodopsin (a chicken red-sensitive cone visual pigment) purified in a detergent system containing CHAPS and phosphatidylcholine was investigated by means of nanosecond laser photolysis at room temperature. Excitation of iodopsin with a nanosecond laser pulse (wavelength, 560 nm; pulse width, 17 ns) resulted in the formation of at least four intermediates on the nanosecond to millisecond time scale. The earliest intermediate detected had an absorption maximum at 571 nm, which was very close to that of original iodopsin (lambda max = 567 nm), and remarkably blue-shifted as compared with that of bathoiodopsin [lambda max = 625 nm; Kandori et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8908-8912]. The intermediate, named BL-iodopsin, converted to the next intermediate, lumiiodopsin (lambda max = 535 nm), with a time constant of 130 ns. The BL intermediate had an absorption maximum just between batho- and lumiiodopsins, and an extinction coefficient comparable with these intermediates. These properties are different from those of the corresponding intermediate of rhodopsin [BL(BSI)-rhodopsin], suggesting that the binding of chloride to iodopsin, but not to rhodopsin, has an influence upon changes of the chromophore-opsin interaction in the early stage of photobleaching of iodopsin. Lumiiodopsin converted to metaiodopsin I (lambda max < 500 nm) with a time constant of 230 microseconds, and then to metaiodopsin II (lambda max = 390 nm) with a time constant of 6 ms. A thermal equilibrium between metaiodopsin I and II was established, but unlike meta intermediates of rhodopsin, they showed little temperature dependence.

Animals↗

PubMed1993

Purification of four forms of the beta gamma subunit complex of G proteins containing different gamma subunits.

To investigate the physiological significance of the diversity of gamma subunits of G proteins, we purified four forms of beta gamma of G proteins from bovine brain (beta gamma-B1, beta gamma-B2, beta gamma-B3), and spleen (beta gamma-S1) by the sequential chromatography on columns of DEAE-Sephacel, Ultrogel AcA 34, heptylamine-Sepharose, phenyl-5PW, and DEAE-5PW. Electrophoretic analyses showed that each beta gamma mainly contained the 36-kDa beta and a distinct but homogeneous gamma. These beta gamma complexes were subjected directly to proteolytic digestion and subsequent amino acid sequence analyses of their fragments. It was revealed that beta gamma-B1, -B2, and -B3 were identical to beta 1 gamma 7 (with a low level of beta 2 gamma 7), beta 1 gamma 2 and beta 1 gamma 3, respectively, while beta gamma-S1 was composed of beta 1 and an unidentified form of gamma. Then we examined the functional differences among these beta gamma complexes and the beta gamma of transducin (beta gamma-T, beta 1 gamma 1). Few differences were observed among all beta gamma complexes to enhance pertussis toxin-catalyzed ADP-ribosylation of the alpha subunits of G(o) and Gt. The four forms of beta gamma complexes purified from brain and spleen showed indistinguishable inhibitory effects on the release of GDP from G(o) alpha, but beta gamma-T was much less effective. Brain and spleen beta gamma complexes were equally effective in inhibiting calmodulin-stimulated adenylyl-cyclase activity, but beta gamma-T had a very weak inhibitory effect. Five forms of beta gamma facilitated metarhodopsin II-catalyzed binding of GTP gamma S to Gt alpha in a concentration-dependent manner with the following rank order of effectiveness: beta gamma-S1 > beta gamma-T > beta gamma-B1 > beta gamma-B2 > beta gamma-B3. Because the beta gamma complexes used in this study mostly contained the same beta subunit, the functional differences must be dependent on the gamma subunits. Thus, it seems likely that the receptor, the alpha subunits, and the effector are able to distinguish between the various gamma subunits.

Adrenal Glands↗

PubMed1993

Activation by G protein beta gamma subunits of beta-adrenergic and muscarinic receptor kinase.

We have shown previously that GTP-binding regulatory protein (G protein) beta gamma subunits stimulate the agonist- or light-dependent phosphorylation of muscarinic acetylcholine receptors (mAChRs) and rhodopsin by a protein kinase partially purified from porcine brain (mAChR kinase) but not the phosphorylation of rhodopsin by rhodopsin kinase (Haga, K., and Haga, T. (1992) J. Biol. Chem. 267, 2222-2227). We report here that the mAChR kinase phosphorylates beta-adrenergic receptors (beta-ARs) purified from bovine lung in an agonist-dependent manner, and the phosphorylation is also stimulated by G protein beta gamma subunits. We also report that recombinant beta-adrenergic receptor kinase 1 (beta-ARK1) expressed in COS-7 cells phosphorylates mAChRs (human m2 subtype) and rhodopsin in an agonist- or light-dependent manner, respectively, and that this phosphorylation is stimulated by G protein beta gamma subunits. By contrast, the beta gamma subunits do not stimulate the phosphorylation of mAChRs or rhodopsin by a beta-ARK1 mutant lacking a part of the carboxyl-terminal region which is present in beta-ARKs but not in rhodopsin kinase. These results indicate that the beta-ARK1 is the same as or very similar to the mAChR kinase but is distinguished from the rhodopsin kinase with respect to activation by the beta gamma subunits and that the extra carboxyl-terminal sequence in beta-ARKs is required for the stimulation by the beta gamma subunits.

Alprenolol↗

PubMed1993

Purification and characterization of three MEKA-like proteins in liver: association of a 94 kDa protein with beta gamma subunits of G-proteins.

Retinal 32 kDa MEKA protein (rMEKA) exists in the photoreceptor cells and forms a complex with beta gamma subunit of transducin. Bovine liver contained three MEKA-like proteins (94 kDa, 35 kDa-a, 35 kDa-b) which reacted with a rMEKA antibody. Each protein was purified as a single band on a SDS-PAGE and used for a reconstitution experiment with alpha and beta gamma subunits of cerebral G-proteins (Go/i). The 94 kDa protein inhibited GTP-binding ability of G alpha by forming a complex with beta gamma subunit.

Animals↗

PubMed1993

Identification of the alpha-subunits of rod and cone transducin in chicken photoreceptor cells.

The chicken retina has several types of cone photoreceptor cells, each of which contains a visual pigment, chicken red (iodopsin), green, blue or violet. Although biochemical and photochemical properties of these cone pigments have been well characterized, no information is available about the chicken photoreceptor G-protein, transducin, which couples with the visual pigment to convert a photon signal into a cellular response. To identify alpha-subunits of chicken rod and cone transducins (Tr alpha and Tc alpha, respectively), we produced two site-directed antibodies which discriminate between bovine Tr alpha and Tc alpha. Immunohistochemical studies on chicken retinas revealed that the antibody against bovine Tr alpha specifically stained the rod outer segments. On the other hand, the antibody against bovine Tc alpha uniformly stained the outer segments of the double cones and all types of single cones, while the single cones were immunohistochemically classified into three types by using a combination of antibodies against bovine rhodopsin and chicken iodopsin. Immuno-blot analyses demonstrated that the antibody against Tc alpha recognized a single band of chicken photoreceptor protein, whose molecular weight (42,000) was in good agreement with that of bovine Tc alpha (41,000). The antibody against Tr alpha recognized a protein having the same molecular weight as that of bovine Tr alpha (39,000). These observations strongly suggested that all types of chicken cone cells have a single common Tc alpha (42 kDa) structurally related to bovine Tc alpha, though each cone cell type has a distinct visual pigment.

Animals↗

PubMed1993

[Photoreceptors and GTP-binding protein in visual cells].

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Adaptation, Ocular↗

PubMed1993

[Light-induced dephosphorylation of phosphoproteins in rod outer segments in bovine and frog retina].

Several lines of evidence have suggested that protein phosphorylation and dephosphorylation may play an important role in the regulation of metabolism and signal transduction processes. In our present study, bovine and frog retinas were incubated in Krebs' solution containing [32P] H3PO4 for the labelling of all phosphoproteins. Then, photoreceptor outer segments were isolated from each retina, and further incubated under dark or light conditions. In such conditions, several phosphoproteins were dark- or light-dependently dephosphorylated. Interestingly, the light-dependent dephosphorylated 39 kDa protein as well as the 35-36 kDa protein was commonly observed in both bovine and frog retinas. The 35-36 kDa protein is considered to be the same as the 33 kDa protein that has previously been shown to be phosphorylated light-dependently, whereas 39 kDa protein is thought to be a novel protein that undergoes light-dependent dephosphorylation in retinal photoreceptor outer segments. Thus, these proteins were thought to have significant roles in the visual transduction processes.

Animals↗

PubMed1992

Identification and isolation of common and tissue-specific geranylgeranylated gamma subunits of guanine-nucleotide-binding regulatory proteins in various tissues.

Heterotrimeric guanine-nucleotide-binding regulatory proteins (G proteins) have been classified into several subtypes on the basis of the properties of their alpha subunits, though a notable multiplicity of gamma subunits has also been demonstrated. To investigate whether each subtype of alpha subunit is associated with a particular gamma subunit, various oligomeric G proteins, purified from bovine tissues, were subjected to gel electrophoresis in a Tricine buffer system. All G proteins examined were shown to have more than two kinds of gamma subunit. Of the brain G proteins, GoA, GoB, and Gi1 contain the same set of three gamma subunits, but Gi2 contains only two of these subunits. Lung Gi1 and Gi2 and spleen Gi2 and Gi3 had similar sets of two gamma subunits, one of which was distinct from the gamma subunits of brain G proteins. These observations indicate that each subtype of alpha subunit is associated with a variety of beta gamma subunits, and that the combinations differ among cells. For analyses of the structural diversity of the gamma subunits, beta gamma subunits were purified from the total G proteins of each tissue and subjected to reverse-phase HPLC under denaturing conditions, where none of the beta subunits were eluted from the column. Three distinct gamma subunits were isolated in this way from brain beta gamma subunits. In contrast, lung and spleen beta gamma subunits contained at least five gamma subunits, the elution positions and electrophoretic mobilities of which were indistinguishable between the two tissues. Among several gamma subunits, two subspecies appeared to be common to the three tissues. In fact, in each case, the partial amino acid sequence of the most abundant gamma subunit in each tissue was identical, and the sequences coincided exactly with that of 'gamma 6' [Robishaw, J. D., Kalman, V. K., Moomaw, C. R. & Slaughter, C. A. (1989) J. Biol. Chem. 264, 15758-15761]. Fast-atom-bombardment mass spectrometry analysis indicated that this abundant gamma subunit in lung and spleen was geranylgeranylated and carboxymethylated at the C-terminus, as was 'gamma 6' from brain. In addition to abundant gamma subunits, other tissue-specific gamma subunits were also shown to be geranylgeranylated by gas-chromatography-coupled mass spectrometry analysis of Raney nickel-treated gamma subunits. These results suggest that most gamma subunits associated with many different subtypes of alpha subunit are geranylgeranylated in a variety of tissues, with the single exception being the retina where the G protein transducin has a farnesylated gamma subunit.

Amino Acid Sequence↗

PubMed1992

Lipid modification at the N terminus of photoreceptor G-protein alpha-subunit.

Myristate is a fatty acid (fourteen-carbon chain with no double bonds, C14:0) linked to the amino-terminal glycine of several proteins, including alpha-subunits of heterotrimeric (alpha/beta gamma) G proteins. We report here a novel modification at the N terminus of the alpha-subunit of the photoreceptor G protein transducin, T alpha, with heterogeneous fatty acids composed of laurate (C12:0), unsaturated C14:2 and C14:1 fatty acids, and a small amount (approximately 5%) of myristate. Both the GTPase activity of T alpha/T beta gamma and the T beta gamma-dependent ADP-ribosylation of T alpha catalysed by pertussis toxin were inhibited by the lauroylated and myristoylated N-terminal peptide of T alpha. The myristoylated peptide gave 50% inhibition at a 3.5 to approximately 4.5-fold lower concentration than the lauroylated peptide in each assay, indicating that the strength of the interaction between T alpha and T beta gamma is altered by heterogeneous fatty acids linked to T alpha. This suggests that a looser subunit interaction in transducin which is due to an abundance of N-linked fatty acids other than myristate would favour the rapid turnover and catalysis essential for the visual excitation in photoreceptor cells.

Adenosine Diphosphate Ribose↗

PubMed1992

Cone visual pigments are present in gecko rod cells.

The Tokay gecko (Gekko gekko), a nocturnal lizard, has two kinds of visual pigments, P467 and P521. In spite of the pure-rod morphology of the photoreceptor cells, the biochemical properties of P521 and P467 resemble those of iodopsin (the chicken red-sensitive cone visual pigment) and rhodopsin, respectively. We have found that the amino acid sequence of P521 deduced from the cDNA was very similar to that of iodopsin. In addition, P467 has the highest homology with the chicken green-sensitive cone visual pigment, although it also has a relatively high homology with rhodopsins. These results give additional strength to the transmutation theory of Walls [Walls, G. L. (1934) Am. J. Ophthalmol. 17, 892-915], who proposed that the rod-shaped photoreceptor cells of lizards have been derived from ancestral cone-like photoreceptors. Apparently amino acid sequences of visual pigments are less changeable than the morphology of the photoreceptor cells in the course of evolution.

Amino Acid Sequence↗

PubMed1992

Primary structures of chicken cone visual pigments: vertebrate rhodopsins have evolved out of cone visual pigments.

The chicken retina contains rhodopsin (a rod visual pigment) and four kinds of cone visual pigments. The primary structures of chicken red (iodopsin) and rhodopsin have been determined previously. Here we report isolation of three cDNA clones encoding additional pigments from a chicken retinal cDNA library. Based on the partial amino acid sequences of the purified chicken visual pigments together with their biochemical and spectral properties, we have identified these clones as encoding the chicken green, blue, and violet visual pigments. Chicken violet was very similar to human blue not only in absorption maximum (chicken violet, 415 nm; human blue, 419 nm) but also in amino acid sequence (80.6% identical). Interestingly, chicken green was more similar (71-75.1%) than any other known cone pigment (42.0-53.7%) to vertebrate rhodopsins. The fourth additional cone pigment, chicken blue, had relatively low similarity (39.3-54.6%) in amino acid sequence to those of the other vertebrate visual pigments. A phylogenetic tree of vertebrate visual pigments constructed on the basis of amino acid identity indicated that an ancestral visual pigment evolved first into four groups (groups L, S, M1, and M2), each of which includes one of the chicken cone pigments, and that group Rh including vertebrate rhodopsins diverged from group M2 later. Thus, it is suggested that the gene for scotopic vision (rhodopsin) has evolved out of that for photopic vision (cone pigments). The divergence of rhodopsin from cone pigments was accompanied by an increase in negative net charge of the pigment.

Amino Acid Sequence↗

PubMed1992

Differentiation of both rod and cone types of photoreceptors in the in vivo and in vitro developing pineal glands of the quail.

The avian pineal is a photo-endocrinal organ and is considered to synthesize and secrete melatonin in an intrapineal rhythm which can be modified by direct light stimulation of the pineal photoreceptors. Since the avian retina contains numerous different types of photoreceptors, at least 6 types in the quail retina, it is interesting to ask how many types of photoreceptors are present in the avian pineal. In the present study, we have identified two types of photoreceptors in the quail pineal organ, one appears rod-like and the other cone-like, using an immunohistochemical method with highly specific anti-chicken rhodopsin and anti-iodopsin monoclonal antibodies. Rhodopsin-immunoreactive (Rho-I) cells were much larger in number than iodopsin-immunoreactive (Iodo-I) cells. During pineal development, Rho-I cells were first observed at embryonic day 13 (E13: 13 days of incubation), whereas Iodo-I cells were found at day E15. Rho-I cells showed numerous neurite-like processes, but Iodo-I cells had few, if any, processes. We developed a new culture system for avian pineal cell differentiation by seeding cells on nitrocellulose membrane filters. By this method both types of pineal photoreceptors differentiated in vitro: Rho-I cells were much larger in number and had much more fine processes than Iodo-I cells, similar to those seen in the intact developing pineal. With the new culture system the relation between pineal photoreceptor differentiation and sympathetic innervation was examined in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Photosensitivities of iodopsin and rhodopsins.

The relative photosensitivity and the molar extinction coefficient of a highly purified iodopsin (chicken red sensitive cone visual pigment) solubilized in a mixture of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and phosphatidylcholine (CHAPS-PC) were measured using bovine rhodopsin solubilized in 2% digitonin as a standard and compared with those of chicken and bovine rhodopsins. The photosensitivity obtained (1.08) was close to those of rhodopsins (chicken, 1.04; bovine, 0.99) in CHAPS-PC. The molar extinction coefficient of iodopsin (47,200) was 1.15-1.17 times higher than those of rhodopsins (chicken, 40,500; bovine, 41,200). The oscillator strength of iodopsin (0.60) calculated from the extinction coefficient was nearly identical to that of chicken rhodopsin (0.61), suggesting that the chromophore of iodopsin is similar in configuration to rhodopsin. In contrast, the difference in quantum yield between iodopsin (0.62) and chicken rhodopsin (0.70) suggests that the chromophore-opsin interaction after absorption of a photon by the chromophore may be different.

Animals↗