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PubMed2004

Temporal kinetics of the light/dark translocation and compartmentation of arrestin and alpha-transducin in mouse photoreceptor cells.

PURPOSE: To determine the temporal kinetics of the simultaneous translocation of arrestin and rod alpha-transducin in mice exposed to different lighting environments and to compare the subcellular compartmentation of cone alpha-transducin with arrestin. METHODS: Double labeling immunofluorescence microscopy and image analysis are used to visualize and quantify the concentrations of rod arrestin and alpha-transducin in the subcellular compartments of the rod outer segments, the rod inner segments and the synaptic terminals. RESULTS: The magnitude of the effects of the translocation are clearly contrasted in images of the retinas of animals that have been maximally light adapted verses retinas that have been maximally dark adapted. The onset of light results in a rapid, simultaneous, translocation of arrestin and alpha-transducin from their respective compartments (alpha-transducin in the rod outer segment and arrestin in the rod inner segment) to the opposite compartment. Almost all of alpha-transducin has translocated in less than two min whereas the translocation of the majority of arrestin requires at least five to six min. Translocation in the opposite direction, from light to dark, occurs more slowly for both proteins with arrestin requiring almost 30 min and alpha-T needing more than 200 min to complete its journey. Under the same lighting conditions, cone arrestin translocation is incomplete. Cone alpha-transducin does not translocate under any the lighting conditions tested. Unlike the frog, continuous exposure of mice to light does not result in arrestin translocating back to the rod inner segment. CONCLUSIONS: These data suggest that there are four mechanisms involved in the translocation of these two proteins. They also support the conclusion that the more important cellular function of the translocation process is to terminate phototransduction in rod and cone photoreceptors, which could provide protection against light damage. The secondary function of translocation is to maximize rod sensitivity to light during dark adaptation. The restricted localization of cone alpha-transducin to the cone outer segment is consistent with the function of cones in bright light, just as the concentration of rod alpha-transducin in dark adapted rod outer segment is consistent with their functioning in dim light.

Animals↗
PubMed1987

Allosteric behavior in transducin activation mediated by rhodopsin. Initial rate analysis of guanine nucleotide exchange.

Photolyzed rhodopsin acts in a catalytic manner to mediate the exchange of GTP for GDP bound to transducin. We have analyzed the steady-state kinetics of this activation process in order to determine the molecular mechanism of interactions between rhodopsin, transducin, and guanine nucleotides. Initial velocities (Vo) of the exchange reaction catalyzed by rhodopsin were measured for various transducin concentrations at several fixed levels of the GTP analog, [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S). The initial rate data analysis rigorously demonstrates that rhodopsin mediates the activation of transducin by a double-displacement catalytic mechanism. The Michaelis-Menten curves determined as a function of [transducin] reveal remarkable allosteric behavior; analysis of this data yields a Hill coefficient of 2. Lineweaver-Burk plots of Vo-1 versus [transducin]-1 display curvilinearity indicative of positive cooperativity and a series of parallel lines are generated by plotting Vo-1 as a function of [transducin]-2. The plots of Vo-1 versus [GTP gamma S]-1 show no evidence of allosterism and are a parallel series. Furthermore, the allosteric behavior observed in the activation of transducin is also witnessed in the rhodopsin-catalyzed guanine nucleotide exchange of the G protein's purified alpha subunit in the absence of the beta X gamma subunit complex. The latter observation implies that the molecular basis for allosterism in the activation process resides in the interactions between the photoreceptor and transducin's alpha subunit.

Allosteric Regulation↗
PubMed1994

Regulation of transducin GTPase activity in bovine rod outer segments.

The photoreceptor G-protein, transducin, belongs to the class of heterotrimeric GTP-binding proteins that transfer information from activated seven-span membrane receptors to effector enzymes or ion channels. Like other G-proteins, transducin acts as a molecular clock. It is activated by photoexcited rhodopsin which catalyzes the exchange of transducin-bound GDP for GTP and then stays active until bound GTP is hydrolyzed by an intrinsic GTPase activity. Our previous study on the components of the amphibian phototransduction cascade (Arshavsky, V. Y., and Bownds, M. D. (1992) Nature 357, 416-417) has shown that transducin GTPase can be significantly accelerated by the target enzyme, cGMP phosphodiesterase (PDE), and more specifically its gamma-subunit (PDE gamma). Here we report that an analogous mechanism is present in bovine photoreceptors. Addition of recombinant PDE gamma to the test photoreceptor membranes which retain transducin but are depleted of endogenous PDE causes a significant acceleration of transducin GTPase activity. A similar effect was observed with the PDE holoenzyme, but not with the complex of PDE alpha- and beta-subunits prepared by a limited proteolysis of PDE with trypsin. The activating effect of PDE gamma is increased as test membrane concentration increases, exceeding 20-fold at rhodopsin concentrations over 80 microM and approaching the rate of the photoresponse turnoff. This suggests either that photoreceptor membranes contain a further factor which is essential for PDE-dependent regulation of transducin-bound GTP hydrolysis or that components of the phototransduction cascade interact in a cooperative manner. We also report that the GTPase-activating epitope is located within the C-terminal third of PDE gamma: the peptide corresponding to the 25 C-terminal amino acid residues of PDE gamma can accelerate transducin GTPase almost as well as the full-length PDE gamma. A part of the GTPase activating epitope is located within the 3 C-terminal amino acid residues: the truncation PDE gamma mutant lacking these residues accelerates transducin GTPase considerably less than the whole length PDE gamma.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1991

Reaction rate and collisional efficiency of the rhodopsin-transducin system in intact retinal rods.

A model of transducin activation is constructed from its partial reactions (formation of metarhodopsin II, association, and dissociation of the rhodopsin-transducin complex). The kinetic equations of the model are solved both numerically and, for small photoactivation, analytically. From data on the partial reactions in vitro, rate and activation energy profile of amplified transducin turnover are modeled and compared with measured light-scattering signals of transducin activation in intact retinal rods. The data leave one free parameter, the rate of association between transducin and rhodopsin. Best fit is achieved for an activation energy of 35 kJ/mol, indicating lateral membrane diffusion of the proteins as its main determinant. The absolute value of the association rate is discussed in terms of the success of collisions to form the catalytic complex. It is greater than 30% for the intact retina and 10 times lower after permeabilization with staphylococcus aureus alpha-toxin. Dissociation rates for micromolar guanosinetriphosphale (GTP) (Kohl, B., and K. P. Hofmann, 1987. Biophys. J. 52:271-277) must be extrapolated linearly up to the millimolar range to explain the rapid transducin turnover in situ. This is interpreted by an unstable rhodopsin-transducin-GTP transient state. At the time of maximal turnover after a flash, the rate of activation is determined as 30, 120, 800, 2,500, and 4,000 activated transducins per photoactivated rhodopsin and second at 5, 10, 20, 30, 37 degrees C, respectively.

Adenosine Diphosphate↗
PubMed1986

Interaction of retinal transducin with guanosine triphosphate analogues: specificity of the gamma-phosphate binding region.

The interaction of six hydrolysis-resistant analogues of GTP with transducin, the signal-coupling protein in vertebrate photoreceptors, was investigated. GppNHp and GppCH2p differ from GTP at the bridging position between the beta- and gamma-phosphate groups. The other analogues studied (GTP gamma F, GTP gamma OMe, GTP gamma OPh, and GTP gamma S) differ from GTP in containing a substituent on the gamma-phosphorus atom or at a nonbridging gamma-oxygen atom. Competition binding experiments were carried out by adding an analogue, [alpha-32P]GTP, and a catalytic amount of photoexcited rhodopsin (R) to transducin and measuring the amount of bound [gamma-32P]GTP. The order of effectiveness of these analogues in binding to transducin was GTP gamma S greater than GTP much greater than GppNHp greater than GTP gamma OPh greater than GTP gamma OMe greater than GppCH2p greater than GTP gamma F A second assay measured the effectiveness of GTP gamma S, GppNHp, and GppCH2p in eluting transducin from disc membranes containing R. The basis of this assay is that transducin is released from disc membranes when it is activated to the GTP form. The relative potency of these three analogues in converting transducin from a membrane-bound to a soluble form was 1000, 75, and 1, respectively. Stimulation of cGMP phosphodiesterase activity served as a third criterion of the interaction of these analogues with transducin. The order of effectiveness of these analogues in promoting the transducin-mediated activation of the phosphodiesterase was GTP gamma S greater than GTP much greater than GppNHp greater than GTP gamma OPh much greater than GppCH2p greater than GTP gamma OMe greater than GTP gamma F GTP gamma S was more than a 1000 times as potent as GTP gamma F in activating the phosphodiesterase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Coupling of bitter receptor to phosphodiesterase through transducin in taste receptor cells.

The rod and cone transducins are specific G proteins originally thought to be present only in photoreceptor cells of the vertebrate retina. Transducins convert light stimulation of photoreceptor opsins into activation of cyclic GMP phosphodiesterase (reviewed in refs. 5-7). A transducin-like G protein, gustducin, has been identified and cloned from rat taste cells. We report here that rod transducin is also present in vertebrate taste cells, where it specifically activates a phosphodiesterase isolated from taste tissue. Furthermore, the bitter compound denatonium in the presence of taste-cell membranes activates transducin but not Gi. A peptide that competitively inhibits rhodopsin activation of transducin also blocks taste-cell membrane activation of transducin, arguing for the involvement of a seven-transmembrane-helix G-protein-coupled receptor. These results suggest that rod transducin transduces bitter taste by coupling taste receptor(s) to taste-cell phosphodiesterase. Phosphodieterase-mediated degradation of cyclic nucleotides may lead to taste-cell depolarization through the recently identified cyclic-nucleotide-suppressible conductance.

Animals↗
PubMed1998

Effect of detergents and lipids on transducin photoactivation by rhodopsin.

Rhodopsin samples, isolated using four different extraction procedures, were used to investigate the photodependent activation of the GTPase activity of transducin. A complete inhibition of transducin light-dependent GTP hydrolytic activity was observed when rhodopsin purified in the presence of 1% digitonin, following rod outer segment (ROS) solubilization with 1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate (CHAPS), was used for its activation [0 pmol of inorganic phosphate (Pi) released/min/pmol of rhodopsin]. Rhodopsin, isolated in the presence of 1% digitonin following ROS solubilization with 1% digitonin, was capable of stimulating slightly transducin GTPase activity, with an initial rate of 1 pmol of GTP hydrolyzed/min/pmol of rhodopsin. However, rhodopsin purified in the presence of 0.2% n-dodecyl-beta-D-maltoside (DM), following ROS solubilization with either 1% CHAPS or 1% DM, stimulated the enzymatic activity of transducin in a light-dependent manner, with an initial rate of 5 pmol of Pi released/min/pmol of rhodopsin. Addition of 0.075% egg phosphatidylcholine (PC) to the four different solubilized rhodopsin samples significantly enhanced light-stimulated GTP hydrolysis by transducin, with initial rates increasing from 0 to 1, 1 to 2, and 5 to 30 pmol of Pi released/min/pmol of rhodopsin, respectively. Furthermore, DM-solubilized rhodopsin induced the hydrolysis of the maximum amount of GTP by transducin at 0.0075% PC, while digitonin-solubilized rhodopsin only stimulated the GTPase activity of transducin to a similar value, when the amount of the photoreceptor protein was increased 4-fold and 0.15% PC was added to the assay mixture. These results suggest that the effective photoactivation of transducin by rhodopsin requires phospholipids, which seem to be differentially eliminated with the detergent extraction procedure utilized during ROS membranes solubilization and photopigment isolation.

Animals↗
PubMed1986

[Inhibitory effect of pertussis toxin on the metabolism of guanine nucleotides in transducin from bovine outer rod segments].

Transducin from bovine retinal rod outer segments possesses two sites responsible for the binding of guanyl nucleotides, one of which is specific only for GTP (GTP-site), while the other one may bind both GTP and GDP (GTP/GDP-site). Pertussis toxin covalently modifies the alpha-subunit of transducin as a result of which 83% of GDP bound at the GTP/GDP site of the protein remain tightly bound and are not displaced by Gpp(NH)p excess. The GTP-site in modified transducin binds Gpp(NH)p at the same rate and reveals the same sensitivity to rhodopsin as does native transducin. Presumably, the GTP/GDP site is localized in the alpha-subunit of transducin. The inhibiting effect of pertussis toxin on GTP hydrolysis by transducin and on stimulation of retinal rod outer segment phosphodiesterase by guanyl nucleotides is due to the tight binding of GDP in the active center of the protein after transducin ADP-ribosylation, which makes impossible the formation of a complex between GTP and the alpha-subunit of transducin.

Animals↗
PubMed1997

Activation of transducin guanosine triphosphatase by two proteins of the RGS family.

RGS proteins (regulators of G protein signaling) constitute a newly appreciated group of negative regulators of G protein signaling. Several members of this group stimulate the guanosine triphosphatase (GTPase) activity of various G protein alpha-subunits, including the photoreceptor G protein, transducin. In photoreceptor cells transducin GTPase is known to be substantially accelerated by the coordinated action of the gamma-subunit of its effector enzyme, cGMP phosphodiesterase (PDE gamma), and another yet unidentified membrane-associated protein factor. Here we test the possibility that this factor belongs to the RGS family of GTPase stimulators. We report a detailed kinetic analysis of transducin GTPase activation by two members of the RGS family, RGS4 and G alpha interacting protein (GAIP). RGS4, being at least 5-fold more potent than GAIP, stimulates the rate of transducin GTPase by 2 orders of magnitude. Neither RGS4 nor GAIP requires PDE gamma for activating transducin. Rather, PDE gamma causes a partial reversal of transducin GTPase activation by RGS proteins. The effect of PDE gamma is based on a decreased apparent affinity of RGS for the alpha-subunit of transducin. Our observations indicate that GTPase activity of transducin can be activated by at least two distinct mechanisms, one based on the action of RGS proteins alone and another involving the cooperative action of the effector enzyme and another protein.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1997

Regulation of transducin GTPase activity by human retinal RGS.

The intrinsic GTPase activity of transducin controls inactivation of the effector enzyme, cGMP phosphodiesterase (PDE), during turnoff of the visual signal. The inhibitory gamma-subunit of PDE (Pgamma), an unidentified membrane factor and a retinal specific member of the RGS family of proteins have been shown to accelerate GTP hydrolysis by transducin. We have expressed a human homologue of murine retinal specific RGS (hRGSr) in Escherichia coli and investigated its role in the regulation of transducin GTPase activity. As other RGS proteins, hRGSr interacted preferentially with a transitional conformation of the transducin alpha-subunit, GtalphaGDPAlF4-, while its binding to GtalphaGTPgammaS or GtalphaGDP was weak. hRGSr and Pgamma did not compete for the interaction with GtalphaGDPAlF4-. Affinity of the Pgamma-GtalphaGDPAlF4- interaction was modestly enhanced by addition of hRGSr, as measured by a fluorescence assay of GtalphaGDPAlF4- binding to Pgamma labeled with 3-(bromoacetyl)-7-diethylaminocoumarin (PgammaBC). Binding of hRGSr to GtalphaGDPAlF4- complexed with PgammaBC resulted in a maximal approximately 40% reduction of BC fluorescence allowing estimation of the hRGSr affinity for GtalphaGDPAlF4- (Kd 35 nM). In a single turnover assay, hRGSr accelerated GTPase activity of transducin reconstituted with the urea-stripped rod outer segment (ROS) membranes by more than 10-fold to a rate of 0.23 s-1. Addition of Pgamma to the reconstituted system reduced the GTPase level accelerated by hRGSr (kcat 0.085 s-1). The GTPase activity of transducin and the PDE inactivation rates in native ROS membranes in the presence of hRGSr were elevated 3-fold or more regardless of the membrane concentrations. In ROS suspensions containing 30 microM rhodopsin these rates exceeded 0.7 s-1. Our data suggest that effects of hRGSr on transducin's GTPase activity are attenuated by Pgamma but independent of a putative membrane GTPase activating protein factor. The rate of transducin GTPase activity in the presence of hRGSr is sufficient to correlate it with in vivo turnoff kinetics of the visual cascade.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1987

Immunological determination of transducin content in retinas exhibiting inherited degeneration.

The inherited disorders of rd mice and affected Irish setter dogs are characterized by the accumulation of cyclic GMP (cGMP). Since the cGMP level in normal retinal rods is regulated by a light-activated enzyme cascade involving rhodopsin, transducin, and phosphodiesterase, an abnormality associated with any of these three proteins would cause cGMP accumulation. In order to determine the relationship between different forms of retinal degeneration and the transducin content in the affected retinas, affinity-purified antibodies directed against the individual subunits of bovine transducin were prepared. These antibodies, which recognized transducin in many vertebrate species, were used to compare the retinal content of this protein at various stages of inherited photoreceptor degeneration. In each of the disorders studied (rd and rds mice, RCS rat, and affected Irish setter dog), retinas at early stages of degeneration displayed two characteristics similar to those of normal control retinas. First, all three subunits of transducin were detected and found to have normal electrophoretic mobility, suggesting that these disorders are unlikely to be due to changes in the composition of transducin subunits. Second, the amount of cross-reactive T beta always exceeded those of T alpha and T gamma. This disproportionately higher amount of T beta-like protein became more pronounced as the visual cells degenerated. In retinas which had undergone complete photoreceptor degeneration, cross-reactive T alpha and T gamma were undetectable. In contrast, anti-T beta gamma antibodies detected an amount of T beta-like polypeptide corresponding to 10-25% of the control. Since our anti-T beta gamma antibodies recognize the beta subunit of the GTP-binding N proteins of the adenylate cyclase system, this finding suggests that this residual T beta-like protein, which is not part of transducin, may be associated with other GTP-binding regulatory proteins.

Animals↗
PubMed1994

Biochemical analysis of the transducin-phosphodiesterase interaction.

In vertebrate rod cells, the activated alpha-subunit of rod transducin interacts with the gamma (regulatory) subunits of phosphodiesterase to disinhibit the catalytic subunits. A 22-amino acid long region of rod transducin involved in phosphodiesterase activation has recently been identified. We have used peptides from this region of rod transducin and from several other G protein alpha-subunits to study the nature and specificity of the G protein alpha-effector interaction. Although peptides derived from rod transducin, cone transducin and gustducin are similar, only the rod peptide is capable of activating rod phosphodiesterase. Using substituted peptides we have identified five residues on one exposed face of rod transducin as important to phosphodiesterase activation. These results disagree with previous models which propose that loop regions of rod transducin interact with phosphodiesterase gamma.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1997

Activation of transducin by a Xenopus short wavelength visual pigment.

Phototransduction in cones differs significantly from that in rods in sensitivity, kinetics, and recovery following exposure to light. The contribution that the visual pigment makes in determining the cone response was investigated biochemically by expressing a Xenopus violet cone opsin (VCOP) cDNA in COS1 cells and assaying the light-dependent activation of transducin. Light-exposed VCOP stimulated [35S]guanosine 5'-(gamma-thio)triphosphate nucleotide exchange on bovine rod transducin in a time-dependent manner with a half-time for activation of 0.75 min, similar to that of bovine rhodopsin. In exhaustive binding assays, VCOP and rhodopsin activity showed similar concentration dependence with half-maximal activation occurring at 0.02 mol of pigment/mol of transducin. Although VCOP was able to activate as many as 12 transducins per photoisomerization, rhodopsin catalyzed significantly more. When assays were performed with lambda > 420 nm illumination, VCOP exhibited rapid regeneration and high affinity for the photoregenerated 11-cis-retinal. Recycling of the chromophore and reactivation of the pigment resulted in multiple activations of transducin, whereas a maximum of 1 transducin per VCOP was activated under brief illumination. The decay of the active species formed following photobleaching was complete in <5 min, approximately 10-fold faster than that of rhodopsin. In vitro, VCOP activated rod transducin with kinetics and affinity similar to those of rhodopsin, but the active conformation decayed more rapidly and the apoprotein regenerated more efficiently with VCOP than with rhodopsin. These properties of the violet pigment may account for much of the difference in response kinetics between rods and cones.

Alcohol Dehydrogenase↗
PubMed1986

[Effect of GDP on transducin interaction with cyclic nucleotide phosphodiesterase and rhodopsin from bovine retinal rods].

In the presence of guanyl nucleotides and rhodopsin-containing retinal rod outer segment membranes, transducin stimulates the light-sensitive cyclic nucleotide phosphodiesterase 5.5-7 times. The activation constant (Ka) for GTP and Gpp(NH)p is 0.25 microM, that for GDP and GDP beta S is 14 and 110 microM, respectively. GDP purified from other nucleotide contaminations at concentrations up to 1 mM does not stimulate phosphodiesterase but binds to transducin and inhibits the Gpp(NH)p-dependent activation of phosphodiesterase. The mode of transducin interaction with bleached rhodopsin also depends on the nature of the bound guanyl nucleotide: in the presence of GDP rhodopsin-containing membranes bind 70-100% of transducin, whereas in the presence of Gpp(NH)p the membranes bind only 13% of the protein. The experimental results suggest that GDP and GTP convert transducin into two different functional states, i.e., the transducin X GTP complex binds to phosphodiesterase causing its stimulation, while the transducin X GDP complex is predominantly bound to rhodopsin.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1984

ADP-ribosylation of transducin by islet-activation protein. Identification of asparagine as the site of ADP-ribosylation.

Islet-activating protein catalyzes the ADP-ribosylation of transducin, a guanine nucleotide-binding regulatory protein that mediates activation of a retinal cyclic GMP-selective phosphodiesterase. Radiolabel from [adenylate-32P]NAD+ was incorporated specifically into the alpha subunit of purified transducin. Maximal levels of incorporation approximated 0.8 mol of ADP-ribose/mol of transducin. A peptide containing the ADP-ribosyl moiety was purified from a tryptic digest of radiolabeled transducin. This peptide was characterized by chemical and enzymatic procedures and by fast atom bombardment mass spectrometry. The primary structure of this peptide was Glu-Asn-Leu-Lys-Asn(ADP-ribose)-Gly-Leu-Phe. It is probable that the peptide originated from the carboxyl terminus of the alpha subunit and that the ADP-ribosyl moiety is attached by an N-glycosidic linkage to the asparagine residue. Transducin associated with retinal disc membranes is also ADP-ribosylated by cholera toxin. Cholera toxin and islet-activating protein sequentially catalyze the incorporation of 1.9 mol of ADP-ribose/mol of transducin, indicating two distinct sites of ADP-ribosylation within transducin.

Adenosine Diphosphate Ribose↗
PubMed1993

Interaction of rhodopsin with the G-protein, transducin.

Rhodopsin, upon activation by light, transduces the photon signal by activation of the G-protein, transducin. The well-studied rhodopsin/transducin system serves as a model for the understanding of signal transduction by the large class of G-protein-coupled receptors. The interactive form of rhodopsin, R*, is conformationally similar or identical to rhodopsin's photolysis intermediate Metarhodopsin II (MII). Formation of MII requires deprotonation of rhodopsin's protonated Schiff base which appears to facilitate some opening of the rhodopsin structure. This allows a change in conformation at rhodopsin's cytoplasmic surface that provides binding sites for transducin. Rhodopsin's 2nd, 3rd and putative 4th cytoplasmic loops bind transducin at sites including transducin's 5 kDa carboxyl-terminal region. Site-specific mutagenesis of rhodopsin is being used to distinguish sites on rhodopsin's surface that are important in binding transducin from those that function in activating transducin. These observations are consistent with and extend studies on the action of other G-protein-coupled receptors and their interactions with their respective G proteins.

Amino Acid Sequence↗
PubMed1997

Phosducin and betagamma-transducin interaction I: effects of post-translational modifications.

The interaction between phosducin and betagamma-transducin plays regulatory roles in light adaptation of photoreceptors. Both phosducin and betagamma-transducin undergo post-translational modifications, with phosducin modified by phosphorylation and the gamma subunit of betagamma-transducin by farnesylation and carboxylmethylation. In this study we exploited the electrophoretic mobilities of these native proteins to develop a micro binding assay and examined the effects of post-translational modifications on binding affinities. It was found that decarboxylmethylation of gamma-transducin increased the mobility of betagamma-transducin during native gel electrophoresis, but decreased the apparent affinity for phosducin by about 2-fold. Phosphorylation of phosducin by protein kinase A increased the mobility but decreased the apparent affinity for betagamma-transducin by at least 3-fold.

Animals↗
PubMed1992

Mono ADP-ribosylation of transducin catalyzed by rod outer segment extract.

Transducin is the retinal rod outer segment (ROS)-specific G protein coupling the photoexcited rhodopsin to cyclic GMP-phosphodiesterase. The alpha subunit of transducin is known to be ADP-ribosylated by bacterial toxins. We investigated the possibility that transducin is modified in vitro by an endogenous ADP-ribosyltransferase activity. By using either ROS, cytosolic extract of ROS or purified transducin in the presence of [alpha-32P]nicotinamide adenine dinucleotide (NAD+), the alpha and beta subunits of transducin were found to be radiolabeled. The labeling was decreased by snake venom phosphodiesterase I (PDE I). The modification was shown to be mono ADP-ribosylation by analyses on thin layer chromatography of the PDE I-hydrolyzed products which revealed only 5'AMP residues. In addition we report that sodium nitroprusside activates the ADP-ribosylation of transducin.

Adenosine Diphosphate↗