RESUMO
Polypeptides of approximately 39, 36 and Assuntos
Células Fotorreceptoras Retinianas Bastonetes/química
, Rodopsina/química
, Roedores
, Transducina/química
, Transducina/isolamento & purificação
, Animais
, Bovinos
, Guanosina Trifosfato/análogos & derivados
, Guanosina Trifosfato/química
, Guanosina Trifosfato/metabolismo
, Guanilil Imidodifosfato/química
, Guanilil Imidodifosfato/metabolismo
, Luz
, Subunidades Proteicas/química
, Subunidades Proteicas/isolamento & purificação
, Subunidades Proteicas/metabolismo
, Células Fotorreceptoras Retinianas Bastonetes/metabolismo
, Rodopsina/metabolismo
, Roedores/metabolismo
, Transducina/metabolismo
RESUMO
Transducin (T), a GTP-binding protein involved in phototransduction of rod photoreceptor cells, is a heterotrimer arranged as two units, the alpha-subunit (T alpha) and the beta gamma-complex (T beta gamma). The role of the carboxyl groups in T was evaluated by labeling with N,N'-dicyclohexylcarbodiimide (DCCD) and 1-ethyl 3-(3-dimethylaminopropyl) carbodiimide (EDC). Only a minor effect on the binding of beta, gamma-imido guanosine 5'-triphosphate (GMPpNp) to T was observed in the presence of the hydrophobic carbodiimide, DCCD. Similarly, the GMPpNp binding activity of the reconstituted holoenzyme was not significantly affected when T alpha was combined with DCCD-treated T beta gamma. However, the binding of guanine nucleotides to the reconstituted T was approximately 50% inhibited when DCCD-labeled T alpha was incubated with T beta gamma. In contrast, treatment of T with the hydrophilic carbodiimide, EDC, completely impaired its GMPpNp-binding ability. EDC-modified T was incapable of interacting with illuminated rhodopsin, as determined by sedimentation experiments. However, rhodopsin only partially protected against the inactivation of T. Additionally, analyses of trypsin digestion patterns showed that fluoroaluminate was not capable of activating the EDC-labeled T sample. The function of the reconstituted holoenzyme was also disrupted when EDC-modified T alpha was combined with T beta gamma, and when EDC-treated T beta gamma was incubated with T alpha.
Assuntos
Dicicloexilcarbodi-Imida/farmacologia , Etildimetilaminopropil Carbodi-Imida/análogos & derivados , Etildimetilaminopropil Carbodi-Imida/farmacologia , Guanilil Imidodifosfato/metabolismo , Rodopsina/efeitos dos fármacos , Segmento Externo da Célula Bastonete/efeitos dos fármacos , Transducina/química , Animais , Bovinos , Concentração de Íons de Hidrogênio , Segmento Externo da Célula Bastonete/química , Transdução de Sinais , Coloração e Rotulagem , Transducina/efeitos dos fármacos , Transducina/metabolismoRESUMO
Transducin (T), a guanine nucleotide binding regulatory protein composed of alpha-, beta-, and gamma-subunits, serves as an intermediary between rhodopsin and cGMP phosphodiesterase during signaling in the visual process. Pyridoxal 5'-phosphate (PLP), a reagent that has been used to modify enzymes that bind phosphorylated substrates, was probed here as an affinity label for T. PLP inhibited the guanine nucleotide binding activity of T in a concentration dependent manner, and was covalently incorporated into the protein in the presence of [3H]NaBH4. Approximately 1 mol of 3H was bound per mol of T. GTP and GTP analogs appreciably hindered the incorporation of 3H to T, suggesting that PLP specifically modified the protein active site. Interestingly, PLP modified both the alpha- and beta-subunits of T. Moreover, PLP in the presence of GDP behaved as a GTP analog, since this mixture was capable of dissociating T from T:photoactivated rhodopsin complexes.
Assuntos
Guanosina Trifosfato/metabolismo , Fosfato de Piridoxal/metabolismo , Coloração e Rotulagem/métodos , Transducina/química , Transducina/metabolismo , Animais , Sítios de Ligação , Boratos/farmacologia , Bovinos , Inibidores Enzimáticos/farmacologia , Olho/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/análogos & derivados , Guanilil Imidodifosfato/antagonistas & inibidores , Guanilil Imidodifosfato/metabolismo , Ligantes , Luz , Fosfatos/farmacologia , Compostos de Potássio/farmacologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/efeitos da radiação , Piridoxal/farmacologia , Fosfato de Piridoxal/farmacologia , TrítioRESUMO
Transducin (T), the G-protein in the visual system, is a heterotrimer arranged as two functional units, Talpha and Tbetagamma. N,N'-1,2-phenylenedimaleimide (o-PDM) and N,N'-1,4-phenylenedimaleimide (p-PDM), two cysteine specific-homobifunctional agents, were used to covalently cross-link T and its units. A complete inhibition in T function was observed in the presence of these compounds. Incubation of Talpha with o-PDM or p-PDM resulted in the formation of high-molecular-weight oligomers of 70-, 105-, 140-, and >200 kDa, as well as intramolecular cross-linked polypeptides that migrated as 35- and 37-kDa bands. Additionally, the treatment of Tbetagamma with both reagents produced a major species of 46-kDa. The combination of intact Talpha and o-PDM- or p-PDM-treated Tbetagamma reconstituted T native activities. On the contrary, when o-PDM- or p-PDM-modified Talpha was incubated with intact Tbetagamma, more than 90% inhibition on T function was observed. Hence, the cysteines modified and/or cross-linked on Talpha represent functionally important residues of T.
Assuntos
Reagentes de Ligações Cruzadas/química , Cisteína/química , Transducina/química , Animais , Bovinos , Cisteína/metabolismo , Guanilil Imidodifosfato/metabolismo , Fenantrolinas/química , Fenilenodiaminas/química , Subunidades Proteicas , Segmento Externo da Célula Bastonete/química , Fatores de Tempo , Transducina/metabolismoRESUMO
The role of transducin sulfhydryl groups was examined by chemical modification with four different reagents: 4-acetamido-4'-maleimidyl-stilbene-2, 2' disulfonic acid (AMDA); 4-vinyl pyridine (VP); 2-nitro-5-thiocyano benzoic acid (NTCBA); and 2, 5-dimethoxystilbene-4'-maleimide (DM). All these compounds rapidly inhibited the [3H]GMPpNp-binding activity of transducin stimulated by photoexcited rhodopsin (R*). Sedimentation experiments showed that the labeling of transducin with AMDA or VP hindered its binding to R* while NTCBA-modified transducin was capable of interacting with the photoreceptor protein. In contrast, DM-labeled transducin precipitated even in the absence of R*. Photoactivated rhodopsin was capable of protecting against the observed AMDA and NTCBA inhibition in transducin function, but not against the inactivation caused by VP or DM. These results suggest the existence of different functional cysteines on transducin that are located in the proximity of the interaction site with the photoreceptor protein, near the guanine nucleotide binding site, or in regions involved in the structural changes taking place upon protein activation. With the use of these reagents, transducin appears to be "frozen" in various conformational stages of its cycle, providing conditions for studying two of the initial steps of the visual process: the light-dependent binding of transducin to rhodopsin and the transducin guanine nucleotide exchange reaction.
Assuntos
Nucleotídeos de Guanina/metabolismo , Rodopsina/metabolismo , Compostos de Sulfidrila/química , Transducina/química , Transducina/metabolismo , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Bovinos , Eletroforese em Gel de Poliacrilamida , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacologia , Nucleotídeos de Guanina/química , Guanilil Imidodifosfato/metabolismo , Luz , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/efeitos da radiação , Conformação Proteica/efeitos dos fármacos , Piridinas/química , Piridinas/farmacologia , Substâncias Redutoras/química , Substâncias Redutoras/farmacologia , Rodopsina/farmacologia , Rodopsina/efeitos da radiação , Segmento Externo da Célula Bastonete/química , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Estilbenos/química , Estilbenos/farmacologia , Reagentes de Sulfidrila/química , Reagentes de Sulfidrila/farmacologia , Ácidos Sulfônicos/química , Ácidos Sulfônicos/farmacologia , Tiocianatos/química , Tiocianatos/farmacologia , Transducina/efeitos dos fármacosRESUMO
Glutamate is to be considered a nociceptive neurotransmitter and glutamatergic antagonists present antinoceptive activity. In this study we investigated the effects of the naturally occurring antinociceptive compounds rutin, geraniin and quercetine extracted from Phyllanthus, as well as the diterpene jatrophone, extracted from Jatropha elliptica on the binding of [3H]glutamate and [3H]GMP-PNP [a GTP analogue which binds to extracellular site(s), modulating the glutamatergic transmission] in rat brain membrane. Jatrophone inhibited [3H]glutamate binding and geraniin inhibited [3H]GMP-PNP binding. Quercetine inhibited the binding of both ligands. These results may indicate a neurochemical parameter possibly related to the antinoceptive activity of these natural compounds.
Assuntos
Córtex Cerebral/metabolismo , Ácido Glutâmico/metabolismo , Guanilil Imidodifosfato/metabolismo , Extratos Vegetais/farmacologia , Plantas Medicinais/química , Animais , Membrana Celular/metabolismo , Ligação Proteica , Ratos , Ratos Wistar , TrítioRESUMO
GMP-PNP, a non-hydrolyzable analog of GTP binds tightly to G-protein in the presence of Mg2+, so that the binding is stable even after exhaustive washings. This property was exploited to prepare membrane samples of rat brain where G-protein GTP-binding sites were saturated with GMP-PNP. Experiments carried out with these membranes showed that GTP, GMP-PNP, GDP-S and GMP (1 mM) inhibit the sodium-independent [3H]glutamate binding by 30-40% [F(4,40) = 5.9; p < .001], whereas only GMP-PNP activates adenylate cyclase activity [F(6,42) = 3.56; p < .01]. The inhibition of sodium-independent [3H]glutamate binding occurred in the absence of Mg2+. These findings suggest that guanine nucleotides may inhibit glutamate binding and activate adenylate cyclase through distinct mechanisms by acting on different sites.
Assuntos
Adenilil Ciclases/metabolismo , Encéfalo/metabolismo , Ácido Glutâmico/metabolismo , Nucleotídeos de Guanina/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Membrana Celular/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Nucleotídeos de Guanina/metabolismo , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacologia , Guanosina Monofosfato/farmacologia , Guanosina Trifosfato/metabolismo , Guanilil Imidodifosfato/metabolismo , Magnésio/farmacologia , Masculino , Ratos , Ratos Wistar , Sódio/farmacologia , Tionucleotídeos/farmacologia , TrítioRESUMO
In G protein-coupled receptors, neurotransmitter-induced binding of GTP to G proteins triggers the activation of effector systems while simultaneously decreasing the affinity of the transmitter for its specific binding site within the receptor-G protein complex. In the present study we show that, in the chick optic tectum, guanine nucleotides inhibit the binding of the glutamate analog, kainate, and activate adenylate cyclase by different mechanisms and acting on different sites. GMP-PNP, a non-hydrolyzable analog of GTP, binds tightly to G proteins so that the binding is stable even after exhaustive washing. By use of this property, we have prepared membrane samples in which G protein GTP-binding sites are pre-saturated with GMP-PNP. Experiments carried out with these membranes show that GMP-PNP, GDP-S and GMP inhibit the binding of [3H]kainate by interacting with site(s) unrelated to G proteins, whereas GMP-PNP activates adenylate cyclase activity by binding to G proteins.