• BMB Reports
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• 제29권4호
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• pp.372-379
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• 1996

Components I and II (CI&II) are major phosphoproteins in the frog rod outer segments (ROS) of retina, whose phosphorylation is light- and cyclic nucleotide-dependent. Although it was reported that CI & II could be chemically cross-linked to ${\beta}{\gamma}-subunit$ of transducin (${\beta}{\gamma}_t$), it was not clear whether CI&II physically interact with ${\beta}{\gamma}_t$, under native conditions. CI&II extracted by hypotonic washing fo ROS membranes showed an overlapped migration with ${\beta}{\gamma}_t$, in sucrose density gradient centrifugation. The elution profile of CI&II in the peripheral membrane fractions from gel filtration chromatography also overlapped that of ${\beta}{\gamma}_t$. These hydrodynamic parameters indicate that the native molecular state of CI&II in the peripheral membrane fraction appears to be within a complex, most likely with ${\beta}{\gamma}_t$. CI&II coeluted with ${\beta}{\gamma}_t$, showed no phosphorylation by endogenous kinase which phosphorylates a serine of CI&II in other fractions. The purified CI&II were not able to inhibit trypsin-activated cGMP-phosphodiesterase, and CI&II were not recognized by a monoclonal antibody against the ${\gamma}-subunit$ of transducin, indicating that CI&II are not y-subunit of PDE or transducin. Thus, it is likely that native CI&II, which undergo a light-dependent phosphorylation/dephosphorylation cycle, can associate with ${\beta}{\gamma}$, in frog photoreceptor membranes, and the complex formation has an inhibitory effect on the endogenous phosphorylation of CI&II.

• Journal of Photoscience
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• 제9권2호
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• pp.275-277
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• 2002

We have recently found that a detergent-resistant raft like membrane (DRM) can be prepared from bovine rod outer segment membranes as a low-density buoyant fraction in sucrose density gradient ultracentrifugation. G protein (transducin) and its effector enzyme (phosphodiesterase: PDE) drastically change their affinities to DRM in the process of phototransduction. We report here that the recruitment of transducin and/or $^2$PDE to DRM has close relationship with their states in signal transduction. Active T$\alpha$/PDE-complex has a high affinity to DRM, whereas inactive transducin, or inactive PDE are excluded from DRM. Active T$\alpha$/PDE-complex seems to bind to a GTPase activating protein (GRS9) in multi- protein complexes localized on DRM. Physiological significance of the multi-protein complex on the raft-like membrane in vertebrate phototransduction would be discussed.

• BMB Reports
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• 제37권2호
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• pp.260-267
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• 2004

Transducin (T), the heterotrimeric guanine nucleotide binding protein in rod outer segments, serves as an intermediary between the receptor protein, rhodopsin, and the effector protein, cGMP phosphodiesterase. Labeling of T with dansyl chloride (DnsCl) inhibited its light-dependent guanine nucleotide binding activity. Conversely, DnsCl had no effect on the functionality of rhodopsin. Approximately 2-3 mol of DnsCl were incorporated per mole of T. Since fluoroaluminate was capable of activating DnsCl-modified T, this lysine-specific labeling compound did not affect the guanine nucleotide-binding pocket of T. However, the labeling of T with DnsCl hindered its binding to photoexcited rhodopsin, as shown by sedimentation experiments. Additionally, rhodopsin completely protected against the DnsCl inactivation of T. These results demonstrated the existence of functional lysines on T that are located in the proximity of the interaction site with the photoreceptor protein.

• Journal of Photoscience
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• 제9권2호
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• pp.44-46
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• 2002

In the vertebrate retina, rods mediate twilight vision and cones daylight vision. Rods have been purified easily from the retina, and thus the phototransduction mechanism in rods is now well documented. However, it has not been possible to purify cones in large quantities, and therefore, the knowledge on the mechanism in cones is limited. Here we report purification of carp (Cyprinus carpio) cones with a stepwise Percoll gradient. Using purified cells, we compared the phototransduction mechanism between rods and cones. The results showed that both transducin activation and phosphodiesterase activation are less effective, and visual pigment phosphorylation is faster in cones. These differences explain lower light-sensitivity and briefer photoresponse time course in cones.

• BMB Reports
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• 제41권7호
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• pp.555-560
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• 2008

The interaction of the rod GTP binding protein, Transducin ($G_t$), with bleached Rhodopsin ($R^*$) was investigated by measuring radiolabeled guanine nucleotide binding to and release from soluble and/or membrane-bound Gt by reconstituting $G_t$ containing bound GDP ($G_t$-GDP) or the hydrolysis-resistant GTP analog guanylyl imidodiphosphate ($G_t$-p[NH]ppG) with $R^*$ under physiological conditions. Release of GDP and p[NH]ppG from $G_t$ occurred to the same extent and with the same light sensitivity both in the presence and absence of added GTP. Significant amounts of $G_t$ without bound nucleotide ($G_{t^-}$) were generated. When ROS containing bleached rhodopsin ($R^*$) were centrifuged in low ionic strength buffer, $G_{t^-}$ remained associated with the membrane fraction, whereas $G_t$-GDP remained in the soluble fraction. These results suggest that $G_t$-GDP and $G_t$-p[NH]ppG have similar affinities for $R^*$. The results also suggest that $G_{t^-}$, rather than $G_t$-GDP, is the moiety which exhibits tight, "light-induced" binding to rhodopsin.

• Reproductive and Developmental Biology
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• 제35권1호
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• pp.99-103
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• 2011

Transducin-like enhancer protein 1(TLE-1) is protein associated with cell proliferation. This study analyzed change of TLE-1 mRNA expression during in vivo and in vitro maturation in porcine oocytes. Oocytes and granulose cells were collected from follicles of <2 mm, 2~6 mm and >6 mm in diameter in slaughtered pig's ovaries. Oocytes collected from follicles of 2~6 mm in diameter were used after in vitro maturation for 0, 10, 20 and 44 h. Cumulus cells and granulose cells were collected after treatment with hyaluronidase. In results, TLE-1 mRNA expression in oocytes collected from follicle >6 mm in diameter is increased, TLE-1 RNA expression in cumulus cells and granulosa cells from follicles <2 mm, 2 mm 6 mm and >6 mm in diameter. However, there is no significant difference. On the other hand, TLE-1 mRNA expression from oocytes cultured for 10 hand 44 h is increased, TLE-1 mRNA in cumulus cells cultured for 10 h is significant increased(p<0.05) than other culture periods. In conclusion, these results show that TLE-1 is expressed in all cell types of oocytes, cumulus cells and granulose cells, and associated with oocyte maturation.

• Journal of Photoscience
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• 제9권2호
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• pp.47-50
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• 2002

Raft is a distinctive membrane domain enriched in a certain class of lipids, cholesterol, and proteins observed on the plasma membrane. Growing evidence has revealed that such membrane domains play key roles in signal transduction, fertilization, development, transmitter release, and so on. Recently, we have isolated raft-like detergent-resistant membrane (DRM) fraction from bovine photoreceptor rod outer segments. Transducin and its effecter, cGMP-phosphodiesterase, elicited stimulus-dependent translocation between detergent-soluble membrane and DRM. This suggested potential importance of such distinct membrane domains in vertebrate phototransduction. Here, we will discuss physiological meaning of the translocation of major components of cGMP cascade to raft-like membrane in phototransduction. We would like to propose a hypothesis that raft-like membrane domains on the disk membrane are the place where cGMP cascade system could be quenched.

• Journal of Photoscience
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• 제7권1호
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• pp.31-34
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• 2000

The heterotrichous ciliate Stentor coeruieus shows a step-up photophobic response to visible light In the previous paper, the existence of GTP-binding proteins was confirmed by using the antisera against the carboxy terminal decapeptide of transducin $\alpha$ subunit. The photoreceptor, stentorin, is localized in the pigment granule. If the immunoreactive G-protein directly interacts with the photoreceptor stentorin, the G-protein expected to be located in the pigment granule rather than plasma membrane. To elucidate the function of the immunoreactive G-protein, the localization of the G-protein in Stentor coeruleus was studied. The results suggest that this G-protein is located in the myoneme involved in the contraction and extension of the cell rather than in the pigment granule.

• Journal of Photoscience
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• 제9권2호
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• pp.246-248
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• 2002

Light is a major environmental signal for entrainment of the circadian clock, but little is known about the phototransduction pathway triggered by light-activation of photoreceptive molecule(s) responsible for the phase shift of the clock in vertebrates. The chicken pineal gland and retina contain the autonomous circadian oscillators together with the photic entrainment pathway, and hence they provide useful experimental model for the clock system. We previously demonstrated the expression and light-dependent activation of rod-type transducin $\alpha$-subunit (Gtl$\alpha$) in the chicken pineal gland. It is unlikely, however, that the pineal Gt$_1$$\alpha$ plays a major role in the photic entrainment, because the light-induced phase shift is unaffected by bloking the signaling function of Gt$_1$$\alpha$. Here, we show the expression of G 11 $\alpha$, an $\alpha$-subunit of another heterotrimeric G-protein, in the chicken pineal gland and retina by cDNA cloning, Northern blot and Western blot analyses. GIl$\alpha$-immunoreactivity was colocalized with pinopsin in the chicken pineal cells and it was found predominantly at the outer segments of photoreceptor cells in the retinal sections, suggesting functional coupling of G11 $\alpha$ with opsins in the both the tissues. By coimmunoprecipitation experiments using the retina, we showed the light- and GTP-dependent interaction between rhodopsin and G11 $\alpha$. Upon ectopic expression of a Gq/ 11-coupled receptor in cultured pineal cells, pharmacological (non-photic) activation of endogenous G11 induced phase-dependent phase shifts of the melatonin rhythm in a manner very similar to the effect of light. These results suggested opsin-G11 pathway contributing to the photic entrainment of the circadian clock.

• Reproductive and Developmental Biology
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• 제33권3호
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• pp.183-187
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• 2009

This study analyzed change of proteins in granulosa cells during the porcine follicuar development by proteomics techniques. Granulosa cells of the follicles, of which the diameter is $2{\sim}4\;mm$ and $6{\sim}10\;mm$, were collected from ovary of slaughtered pig that each follicle of diameter $1{\sim}4\;mm$ and $6{\sim}10\;mm$. We extracted glanulosa cell proteins by M-PER Mammalian Protein Extraction Reagent. Proteins were refined by clean-up kit and quantified by Bradford method until total protein was $200{\mu}l$. Immobilized pH gradient(IPG) strip used 18 cm, $3{\sim}10\;NL$. SDS-PAGE used 10% acrylamide gel. After silver staining, Melanie 7 and naked eye test were used for spot analyzation. Increasing proteins in glanulosa cell of $6{\sim}10\;mm$ follicle were 7 spots. This spots were analyzed by MALDI-TOF MS and searched on NCBInr. In results, 7 spots were similar to zinc/ling finger protein 3 precursor (RING finger protein 203), angiomotin, heat shock 60 kDa protein 1 (chaperonin) isoform 1 (HSP60), similar to transducin-like enhancer protein 1 (TLE 1), SH3 and PX domains 2A (SH3PXD2A). Those proteins were related with transfer between cells. Increase of proteins has an effect on follicular development.