RESUMEN
The effect of twinfilin-1 on the structure and dynamics of monomeric actin was investigated with fluorescence spectroscopy and differential scanning calorimetry experiments. Fluorescence anisotropy measurements proved that G-actin and twinfilin-1 could form a complex. Due to the formation of the complexes the dissociation of the nucleotide slowed down from the nucleotide-binding pocket of actin. Fluorescence quenching experiments showed that the accessibility of the actin bound ε-ATP decreased in the presence of twinfilin-1. Temperature dependent fluorescence resonance energy transfer and differential scanning calorimetry experiments revealed that the protein matrix of actin becomes more rigid and more heat resistant in the presence of twinfilin-1. The results suggest that the nucleotide binding cleft shifted into a more closed and stable conformational state of actin in the presence of twinfilin-1.
Asunto(s)
Actinas/química , Proteínas de Microfilamentos/metabolismo , Animales , Etenoadenosina Trifosfato/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Ratones , Espectrometría de Fluorescencia , TemperaturaRESUMEN
The vacuolar ATPase enzyme complex (V-ATPase) pumps protons across membranes, energized by hydrolysis of ATP. Extensive investigations on structural and biochemical features of these molecules have implied their importance in the physiological process. In this study, a full-length sequence encoding a vacuolar ATP synthase subunit ε-like protein of Clonorchis sinensis (CsATP-ε) was isolated from our cDNA library. The hypothetical 226 amino acid sequence shared 76% identity with ATP-ε proteins of Schistosoma japonicum and above 55% identity with ATP-ε proteins from human and other eukaryotes. Characteristic Asp140 amino acid residues and seven B-cell epitopes were predicted in this sequence. The complete coding sequence of the gene was expressed in Escherichia coli. Recombinant CsATP-ε (rCsATP-ε) protein could be probed by anti-rCsATP-ε rat serum and C.sinensis-infected human serum in Western blotting experiment, indicating that it is an antigen of strong antigenicity. The high level of antibody titers (1:204,800) showed that CsATP-ε has a powerful immunogenicity. Both the increased level and the change trend of IgG1/IgG2a subtypes in serum showed that the rCsATP-ε can induce strong combined Th1/Th2 immune responses in rats and stimulate the immune response changes to the dominant Th2 from Th1 along with long time infection. The results of immunoblot and immunolocalization demonstrated that CsATP-ε was consecutively expressed at various developmental stages of the parasite, which was supported by real-time PCR analysis. In immunohistochemistry, CsATP-ε was localized on the intestine, vitellarium, and testicle of an adult worm and excretory bladder of metacercaria, implying that CsATP-ε may relate to energy intake and metabolism. This fundamental study would contribute to further researches that are related to growth and development and immunomodulation of C. sinensis.
Asunto(s)
Clonorchis sinensis/enzimología , Etenoadenosina Trifosfato/inmunología , ATPasas de Translocación de Protón Vacuolares/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antihelmínticos/sangre , Clonación Molecular , Clonorchis sinensis/genética , Clonorchis sinensis/inmunología , Epítopos de Linfocito B/inmunología , Humanos , Inmunidad Celular , Inmunoglobulina G/sangre , Datos de Secuencia Molecular , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
Although the number of papers about "vanadium" has doubled in the last decade, the studies about "vanadium and actin" are scarce. In the present review, the effects of vanadyl, vanadate and decavanadate on actin structure and function are compared. Decavanadate (51)V NMR signals, at -516 ppm, broadened and decreased in intensity upon actin titration, whereas no effects were observed for vanadate monomers, at -560 ppm. Decavanadate is the only species inducing actin cysteine oxidation and vanadyl formation, both processes being prevented by the natural ligand of the protein, ATP. Vanadyl titration with monomeric actin (G-actin), analysed by EPR spectroscopy, reveals a 1:1 binding stoichiometry and a K(d) of 7.5 µM(-1). Both decavanadate and vanadyl inhibited G-actin polymerization into actin filaments (F-actin), with a IC(50) of 68 and 300 µM, respectively, as analysed by light scattering assays, whereas no effects were detected for vanadate up to 2 mM. However, only vanadyl (up to 200 µM) induces 100% of G-actin intrinsic fluorescence quenching, whereas decavanadate shows an opposite effect, which suggests the presence of vanadyl high affinity actin binding sites. Decavanadate increases (2.6-fold) the actin hydrophobic surface, evaluated using the ANSA probe, whereas vanadyl decreases it (15%). Both vanadium species increased the ε-ATP exchange rate (k = 6.5 × 10(-3) s(-1) and 4.47 × 10(-3) s(-1) for decavanadate and vanadyl, respectively). Finally, (1)H NMR spectra of G-actin treated with 0.1 mM decavanadate clearly indicate that major alterations occur in protein structure, which are much less visible in the presence of ATP, confirming the preventive effect of the nucleotide on the decavanadate interaction with the protein. Putting it all together, it is suggested that actin, which is involved in many cellular processes, might be a potential target not only for decavanadate but above all for vanadyl. By affecting actin structure and function, vanadium can regulate many cellular processes of great physiological significance.
Asunto(s)
Actinas/química , Actinas/metabolismo , Vanadatos/química , Cisteína/química , Etenoadenosina Trifosfato/metabolismo , Resonancia Magnética Nuclear Biomolecular , Oxidación-ReducciónRESUMEN
Actin depolymerizing factor (ADF)/cofilin and profilin are small actin-binding proteins, which have central roles in cytoskeletal dynamics in all eukaryotes. When bound to an actin monomer, ADF/cofilins inhibit the nucleotide exchange, whereas most profilins accelerate the nucleotide exchange on actin monomers. In this study the effects of ADF/cofilin and profilin on the accessibility of the actin monomer's ATP-binding pocket was investigated by a fluorescence spectroscopic method. The fluorescence of the actin bound epsilon-ATP was quenched with a neutral quencher (acrylamide) in steady-state and time dependent experiments, and the data were analyzed with a complex form of the Stern-Volmer equation. The experiments revealed that in the presence of ADF/cofilin the accessibility of the bound epsilon-ATP decreased, indicating a closed and more compact ATP-binding pocket induced by the binding of ADF/cofilin. In the presence of profilin the accessibility of the bound epsilon-ATP increased, indicating a more open and approachable protein matrix around the ATP-binding pocket. The results of the fluorescence quenching experiments support a structural mechanism regarding the regulation of the nucleotide exchange on actin monomers by ADF/cofilin and profilin.
Asunto(s)
Factores Despolimerizantes de la Actina/metabolismo , Actinas/química , Actinas/metabolismo , Adenosina Trifosfato/metabolismo , Profilinas/metabolismo , Factores Despolimerizantes de la Actina/química , Adenosina Trifosfato/química , Algoritmos , Animales , Sitios de Unión , Etenoadenosina Trifosfato , Proteínas Fúngicas/metabolismo , Profilinas/química , Conejos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Espectrometría de FluorescenciaRESUMEN
Cofilin plays an important role in actin turnover in cells by severing actin filaments and accelerating their depolymerization. The role of pH in the severing by cofilin was examined using fluorescence microscopy. To facilitate the imaging of actin filaments and to avoid the use of rhodamine phalloidin, which competes with cofilin, alpha-actin was labeled with tetramethylrhodamine cadaverine (TRC) at Gln41. The TRC-labeling inhibited actin treadmilling strongly, as measured by epsilonATP release. Cofilin binding, detected via an increase in light scattering, and the subsequent conformational change in filament structure, as detected by TRC fluorescence decay, occurred 2-3 times faster at pH 6.8 than at pH 8.0. In contrast, actin filaments severing by cofilin was pH-independent. The pH-independent severing by cofilin was confirmed using actin labeled at Cys374 with Oregon Green 488 maleimide. The depolymerization of actin by cofilin was faster at high pH.
Asunto(s)
Factores Despolimerizantes de la Actina/metabolismo , Actinas/metabolismo , Saccharomyces cerevisiae/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Ácidos Carboxílicos/química , Etenoadenosina Trifosfato/metabolismo , Concentración de Iones de Hidrógeno , Ratones , Unión Proteica , Conejos , Rodaminas/química , Saccharomyces cerevisiae/citología , Factores de TiempoRESUMEN
1. The aim of the present study was to compare ecto-nucleotidase activities in rat bisected vas deferens using 1,N6-etheno(epsilon)-nucleotides (epsilon-ATP and epsilon-AMP) as substrates. Degradation was estimated by measuring the disappearance of the substrate and the appearance of its metabolites using HPLC with fluorescence detection. Incubation of tissue preparations (prostatic or epididymal portions) with 300 nmol/L epsilon-ATP at 37 degrees C caused a partial disappearance of epsilon-ATP and appearance of its metabolites (epsilon-ADP, epsilon-AMP and epsilon-adenosine). Incubation at 25 degrees C reduced epsilon-ATP degradation more in the prostatic than in the epididymal portion. 2. Incubation of tissue preparations with epsilon-AMP at 37 degrees C resulted in the disappearance of epsilon-AMP and the appearance of epsilon-adenosine, which was more pronounced in the epididymal than in the prostatic portion. Incubation at 25 degrees C reduced epsilon-AMP degradation more in the epididymal than in the prostatic portion. 3. Decreasing pH from 7.4 to 6.5 enhanced epsilon-AMP degradation only in the prostatic portion, whereas increasing pH from 7.4 to 8.5 enhanced epsilon-AMP degradation in both portions, but more markedly in the epididymal portion. The alkaline phosphatase inhibitors levamisole (10 mmol/L) and beta-glycerophosphate (10 mmol/L) reduced epsilon-AMP degradation only in the epididymal portion. 4. In conclusion, the results of the present study are compatible with the presence, in the bisected rat vas deferens, of an ecto-nucleotidase system that is involved in the degradation of extracellular purines, which may differ between the epididymal and prostatic portions, with the epididymal portion presenting a different and higher capacity to form adenosine.
Asunto(s)
Nucleótidos de Adenina/metabolismo , Adenosina/metabolismo , Conducto Deferente/metabolismo , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/metabolismo , Animales , Epidídimo/metabolismo , Etenoadenosina Trifosfato/metabolismo , Concentración de Iones de Hidrógeno , Masculino , Próstata/metabolismo , Ratas , Ratas Wistar , Temperatura , Conducto Deferente/anatomía & histologíaRESUMEN
Despite the significance of the elucidation of proteins' physicochemical parameters to understand various molecular phenomena, direct methods for measuring these parameters are not readily available. Here, we propose the use of 8-[p-amino-Ph]-epsilon-ATP, 3b, as a fluorescent probe for the elucidation of physicochemical parameters of binding sites in certain proteins. We synthesized novel fluorescent nucleotide analogues based on an extension of the epsilon-ATP scaffold. These analogues bear a primary or tertiary p-amino-phenyl moiety on the etheno-bridge. We explored the recognition of the fluorescent analogues by the target proteins: P2Y(1)-receptor (P2Y(1)-R) and NTPDase1. Based on the high affinity to the P2Y(1)-R (EC(50) 100nM), 3b proved a suitable probe for the investigation of this receptor. Next, we elucidated the dependencies of the absorption and emission spectra of 3b on environmental parameters, for establishing correlation equations. These equations will help determine the properties of the ATP-binding site from the spectral data of the protein-bound 3b. For this purpose, the sensitivity of the probe to acidity, dielectricity, H-bonding, viscosity, and to correlation between these parameters was determined. Thus, the pH-dependence of 3b emission intensity is bell shaped. At pH2.8 the quantum yield (phi) is enhanced 150-fold, as compared to neutral pH. The basic nitrogen atoms of 3b were assigned and pK(a) values were determined. A linear relationship was found between log phi and log viscosity, however, emission maxima (lambda(max)) remained constant. A linear relationship was found between both phi and lambda(max) and dielectricity, as measured in protic or aprotic solvents of comparable viscosity. pK(a)-like values were measured in acid-titrated alcohols with varying dielectricity but comparable viscosity, or with varying viscosity but comparable dielectricity. An inverse relationship and a linear relationship were found between the pK(a) values of 3b and the medium dielectricity and viscosity, respectively. These correlations help the calibration of properties of a protein ATP-binding site.
Asunto(s)
Etenoadenosina Trifosfato/análogos & derivados , Colorantes Fluorescentes/síntesis química , Proteínas/química , Adenosina Trifosfatasas/química , Antígenos CD/química , Apirasa/química , Sitios de Unión , Etenoadenosina Trifosfato/síntesis química , Fluorescencia , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Espectrometría de Fluorescencia , Electricidad Estática , ViscosidadRESUMEN
Two fluorescent adenosine derivatives (5 and 7) (Sch. 1) and two 6-amino-9-ethylpurine derivatives (6 and 8) (Sch. 1), were synthesised using 2-chloropropanal and 3-chloropropyne as reagents. The structures of the products were determined by spectroscopic and spectrometric methods (1H-, 13C- and 2D NMR, MS, UV and fluorescence spectrometry). Their fluorescence properties were determined and found to be similar to those of ethenoadenosine. Also, the stabilites of 5 and 7 in aqueous solutions were determined and found to be higher than that of the etheno derivative of adenosine.
Asunto(s)
Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Adenosina/análogos & derivados , Etenoadenosina Trifosfato/química , Estructura Molecular , Nucleósidos/química , Purinas/química , Espectrometría de FluorescenciaRESUMEN
The interaction of profilin and non-muscle beta,gamma-actin prepared from bovine spleen has been investigated under physiologic ionic conditions. Profilin binding to actin decreases the affinity of actin for MgADP and MgATP by about 65- and 13-fold, respectively. Kinetic measurements indicate that profilin binding to actin weakens the affinity of actin for nucleotides primarily due to an increased nucleotide dissociation rate constant, but the nucleotide association rate constant is also increased about 2-fold. Removal of the actin-bound nucleotide and divalent cation produces the labile intermediate species in the nucleotide exchange reaction, nucleotide free actin (NF-actin), and increases the affinity of actin for profilin about 10-fold. Profilin binds NF-actin with high affinity, K(D) = 0.013 microM, and slows the observed denaturation rate of NF-actin. Addition of ATP to NF-actin weakens the affinity for profilin and addition of Mg(2+) to ATP-actin further weakens the affinity for profilin. The high-affinity Mg(2+) of actin regulates binding of both nucleotide and profilin to actin and is important for actin interdomain coupling. The data suggest that profilin binding to actin weakens nucleotide binding to actin by disrupting Mg(2+) coordination in the actin central cleft.
Asunto(s)
Actinas/metabolismo , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Contráctiles , Magnesio/metabolismo , Proteínas de Microfilamentos/metabolismo , Animales , Sitios de Unión , Cationes Bivalentes/metabolismo , Bovinos , Etenoadenosina Trifosfato/metabolismo , Cinética , Modelos Químicos , Profilinas , Desnaturalización Proteica , Isoformas de Proteínas/metabolismo , Espectrometría de Fluorescencia , Termodinámica , Triptófano/químicaRESUMEN
ATP transport to synaptic vesicles from rat brain has been studied using the fluorescent substrate analogue 1,N6-ethenoadenosine 5'-triphosphate (epsilon-ATP). The increase in intravesicular concentration was time dependent for the first 30 min, epsilon-ATP being the most abundant nucleotide. The complexity of the saturation curve indicates the existence of kinetic and allosteric cooperativity in the nucleotide transport, which exhibits various affinity states with K0.5 values of 0.39 +/- 0.06 and 3.8 +/- 0.1 mM with epsilon-ATP as substrate. The Vmax values obtained were 13.5 +/- 1.4 pmol x min(-1) x mg of protein(-1) for the first curve and 28.3 +/- 1.6 pmol x min(-1) x mg of protein(-1) considering both components. This kinetic behavior can be explained on the basis of a mnemonic model. The nonhydrolyzable adenine nucleotide analogues adenosine 5'-O-3-(thiotriphosphate), adenosine 5'-O-2-(thiodiphosphate), and adenosine 5'-(beta,gamma-imino)triphosphate and the diadenosine polyphosphates P1,P3-di(adenosine)triphosphate, P1,P4-di(adenosine)tetraphosphate, and P1,P5-di(adenosine)pentaphosphate inhibited the nucleotide transport. The mitochondrial ATP/ADP exchange inhibitor atractyloside, N-ethylmaleimide, and polysulfonic aromatic compounds such as Evans blue and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid also inhibit epsilon-ATP vesicular transport.
Asunto(s)
Adenosina Trifosfato/metabolismo , Encéfalo/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Transporte Biológico/fisiología , Cromatografía Líquida de Alta Presión , Etenoadenosina Trifosfato/metabolismo , Colorantes Fluorescentes/metabolismo , Técnicas In Vitro , Cinética , Masculino , Ratas , Ratas WistarRESUMEN
Alkylation of myosin's Cys-707 (SH1) and Cys-697 (SH2) has profound consequences for myosin's ability to interact with actin and hydrolyze MgATP. Pre-steady-state measurements of myosin-S1 alkylated at SH1 and SH2 by N-phenylmaleimide (NPM) in the presence of ATP were taken to identify the steps of the reaction that are altered. It was found that the rate constant most affected by this modification is the apparent rate of the ATP hydrolysis step. This rate constant is reduced 20000-fold, an effect comparable in magnitude to the effect of the same modification on the binding of MgATP to S1 or acto-S1 [Xie, L., and Schoenberg, M. (1998) Biochemistry 37, 8048]. In contrast, the rate constants of phosphate release and dissociation of acto-S1 by ATP were reduced <20-fold. For unmodified S1, the enhancement of fluorescence seen after addition of ATP had the same rate constant as the ATP hydrolysis step (S1.ATP if S1.ADP.Pi) measured by single-turnover experiments in a quench-flow experiment. This is consistent with results previously observed [Johnson, K. A., and Taylor, E. W. (1978) Biochemistry 17, 3432]. However, NPM-modified S1 exhibited virtually no fluorescence enhancement upon ATP binding. This provides further evidence that M.ATP is the predominant intermediate of NPM-S1-catalyzed ATP hydrolysis.
Asunto(s)
Maleimidas/química , Subfragmentos de Miosina/química , Subfragmentos de Miosina/metabolismo , Actinas/química , Actinas/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión , Etenoadenosina Trifosfato/análogos & derivados , Etenoadenosina Trifosfato/química , Etenoadenosina Trifosfato/metabolismo , Hidrólisis , Cinética , Conejos , Espectrometría de Fluorescencia , EspectrofotometríaRESUMEN
Flow cytometry techniques, usually employed to characterize cellular populations, are reported here to be a valuable tool to approach the study of subcellular organelle functioning. Chromaffin granules rendered fluorescent by using an antibody against their membrane protein, synaptophysin, are detectable by flow cytometry. Moreover, these storage granules are able to transport the fluorescent ATP analogue, epsilon-ATP (1,N6-ethenoadenosine 5'-triphosphate), and the resulting granular fluorescence increase can also be followed by this technique. The saturation studies show a non-hyperbolic kinetic behaviour, with a two step curve. The K0.5 values were 0.26 and 2.5 mM and Hill numbers 1 and 6 respectively. In addition, an unexpected granular size increase, which was dependent on the epsilon-ATP concentration, occurred together with the fluorescence increase. Other nucleotide triphosphate substrates of V-ATPase, such as ATP or GTP, but not the non-hydrolyzable analogue ATP gamma S (adenosine 5'-O-(3-thiotriphosphate), mimic this effect, which exhibited sigmoidal saturation curves with K0.5 values of 1.8 and 3.1 mM for ATP and epsilon-ATP respectively. The V-ATPase inhibitors, suramin, EGTA or EDTA significantly reduced the granular size increase in the presence of ATP. Extragranular addition of noradrenaline has no effect by itself on the granular size, but significantly reduced the granular size increase induced by ATP. This effect was reversed by the amine transport inhibitor reserpine. The granular size increase induced by ATP was more effective in the presence of Cl- than Br- or I-. Moreover, no increase occurred in the presence of F- or acetate. The Cl- channel blockers were poorly effective, and only 2-(phenylamino)-benzoic acid (DPC) exhibited an effect on the ATP-induced granular size increase.
Asunto(s)
Glándulas Suprarrenales , Gránulos Cromafines/metabolismo , Etenoadenosina Trifosfato/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Cationes Monovalentes/farmacología , Bovinos , Canales de Cloruro/antagonistas & inhibidores , Gránulos Cromafines/efectos de los fármacos , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Norepinefrina/farmacología , Tamaño de la Partícula , Reserpina/farmacología , Sinaptofisina/aislamiento & purificaciónRESUMEN
The structure of profilin from the budding yeast Saccharomyces cerevisiae has been determined by X-ray crystallography at 2.3 A resolution. The overall fold of yeast profilin is similar to the fold observed for other profilin structures. The interactions of yeast and human platelet profilins with rabbit skeletal muscle actin were characterized by titration microcalorimetry, fluorescence titrations, and nucleotide exchange kinetics. The affinity of yeast profilin for rabbit actin (2.9 microM) is approximately 30-fold weaker than the affinity of human platelet profilin for rabbit actin (0.1 microM), and the relative contributions of entropic and enthalpic terms to the overall free energy of binding are different for the two profilins. The titration of pyrene-labeled rabbit skeletal actin with human profilin yielded a Kd of 2.8 microM, similar to the Kd of 2.0 microM for the interaction between yeast profilin and pyrene-labeled yeast actin. The binding data are discussed in the context of the known crystal structures of profilin and actin, and the residues present at the actin-profilin interface. The affinity of yeast profilin for poly-L-proline was determined from fluorescence measurements and is similar to the reported affinity of Acanthamoeba profilin for poly-L-proline. Yeast profilin was shown to catalyze adenine nucleotide exchange from yeast actin almost 2 orders of magnitude less efficiently than human profilin and rabbit skeletal muscle actin. The in vivo and in vitro properties of yeast profilin mutants with altered poly-L-proline and actin binding sites are discussed in the context of the crystal structure.
Asunto(s)
Proteínas Contráctiles , Proteínas Fúngicas/química , Proteínas de Microfilamentos/química , Saccharomyces cerevisiae/química , Actinas/química , Actinas/metabolismo , Animales , Bovinos , Cristalografía por Rayos X , Etenoadenosina Trifosfato/metabolismo , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Humanos , Cinética , Proteínas de Microfilamentos/aislamiento & purificación , Proteínas de Microfilamentos/metabolismo , Modelos Moleculares , Péptidos/metabolismo , Profilinas , Unión Proteica , Desnaturalización Proteica , Pliegue de Proteína , Conejos , Termodinámica , UreaRESUMEN
Properties of human profilin I mutated in the major actin-binding site were studied and compared with wild-type profilin using beta/gamma-actin as interaction partner. The mutants ranged in affinity, from those that only weakly affected polymerization of actin to one that bound actin more strongly than wild-type profilin. With profilins, whose sequestering activity was low, the concentration of free actin monomers observed at steady-state of polymerization [Afree], was close to that seen with actin alone ([Acc], critical concentration of polymerization). Profilin mutants binding actin with an intermediate affinity like wild-type profilin caused a lowering of [Afree] as compared to [Acc], indicating that actin monomers and profilin:actin complexes participate in polymer formation. With a mutant profilin, which bound actin more strongly than the wild-type protein, an efficient sequestration of actin was observed, and in this case, the [Afree] at steady state was again close to [Acc], suggesting that the mutant profilin:actin had a greatly lowered ability to incorporate actin subunits at the (+)-end. The results from the kinetic and steady-state experiments presented are consonant with the idea that profilin:actin complexes are directly incorporated at the (+)-end of actively polymerizing actin filaments, while they do not support the view that profilin facilitates polymer formation.
Asunto(s)
Actinas/metabolismo , Proteínas Contráctiles , Etenoadenosina Trifosfato/metabolismo , Proteínas de Microfilamentos/metabolismo , Polímeros/metabolismo , Actinas/química , Animales , Bovinos , Humanos , Cinética , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/aislamiento & purificación , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Profilinas , Unión Proteica , Relación Estructura-ActividadRESUMEN
The H4-H5 loop of the alpha-subunit of mouse brain Na,K-ATPase was expressed and isolated from Escherichia coli cells. Using fluorescence analogues of ATP, this loop was shown to retain its capability to bind ATP. Isolation of a soluble H4-H5 loop with the native ATP binding site is a crucial step for detailed studies of the molecular mechanism of ATP binding and utilisation.
Asunto(s)
Adenosina Trifosfato/metabolismo , Citoplasma/enzimología , Fragmentos de Péptidos/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Sitios de Unión/genética , Encéfalo , Escherichia coli/enzimología , Escherichia coli/genética , Etenoadenosina Trifosfato/metabolismo , Vectores Genéticos/síntesis química , Ratones , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/aislamiento & purificación , ATPasa Intercambiadora de Sodio-Potasio/biosíntesisRESUMEN
Efficient control of synaptic transmission requires a rapid mechanism for terminating the actions of neurotransmitters. For amino acids and monoamines, this is achieved by their uptake into the cell by specific high-affinity transporters; acetylcholine is first broken down in the extracellular space and then choline is taken up by the cell. Because ATP is hydrolysed to adenosine by membrane-bound enzymes (ectonucleotidases) that are present in most tissues, it has been assumed that these enzymes terminate the neurotransmitter actions of ATP in the brain and in the periphery. We show here, however, that stimulation of sympathetic nerves innervating the guinea-pig vas deferens releases not only neuronal ATP, but also soluble nucleotidases that break down this ATP to adenosine, indicating that inactivation of ATP is increased by nerve activity. This release of specific nucleotidases together with ATP represents a new mechanism for terminating the actions of a neurotransmitter.
Asunto(s)
Neuronas/metabolismo , Neurotransmisores/metabolismo , Nucleotidasas/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Animales , Calcio/metabolismo , Cromatografía Líquida de Alta Presión , Inhibidores Enzimáticos/farmacología , Etenoadenosina Trifosfato/metabolismo , Cobayas , Técnicas In Vitro , Masculino , Neurotransmisores/antagonistas & inhibidores , Norepinefrina/metabolismo , Nucleotidasas/antagonistas & inhibidores , Solubilidad , Sistema Nervioso Simpático/citología , Sistema Nervioso Simpático/metabolismo , Conducto Deferente/inervación , Conducto Deferente/metabolismoRESUMEN
The kinetics of ATP exchange on subtilisin-cleaved G-actin was investigated by measuring the fluorescence of 1,N6-ethenoadenosine 5'-triphosphate. The apparent dissociation rate of ATP (k-ATP) was 2.8-fold larger than that of intact G-actin in the presence of 300 microM free Ca2+. Analysis of the dependence of k-ATP on free Ca2+ showed that the dissociation rate constant of tightly bound Ca2+ was not significantly changed by subtilisin cleavage. On the other hand, an equilibrium binding study using 8-amino-2-[(2-amino-5-methylphenoxy)-methyl]-6-methoxyquinoline N,N,N',N'-tetraacetic acid (Quin 2) showed that the affinity of tightly bound Ca2+ for G-actin was reduced by about 13-fold after subtilisin treatment. Consequently, the stabilization by Ca2+ of ATP was weak in cleaved G-actin. Furthermore, the kinetic analysis of ATP exchange revealed that the binding equilibrium between ATP and divalent cation-free cleaved G-actin was much slower than that in the case of intact G-actin.
Asunto(s)
Actinas/metabolismo , ADN/metabolismo , Subtilisinas/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Calcio/metabolismo , Etenoadenosina Trifosfato/metabolismo , Cinética , ConejosRESUMEN
Adenosine 5'-tetraphosphate (Ap4) is a natural constituent of chromaffin granules with concentration values of 2.2 +/- 0.1 nmol/mg of protein and a ratio 245 +/- 40 times lower with respect to ATP (n = 4). The granular transport of epsilon-ATP resulted in a time- and concentration-dependent production of epsilon-adenosine tetraphosphate (epsilon-Ap4) at the intragranular level. The epsilon-Ap4 formation followed a hyperbolic saturation kinetic at low epsilon-ATP concentrations with K(m) value of 0.4 microM epsilon-ATP intragranular (1.15 pmol/mg of granular protein). Intragranular concentrations of epsilon-ATP higher than 500 pmol/mg of protein (approximately to 175 microM intragranular) resulted in a non-saturable production of epsilon-Ap4.
Asunto(s)
Nucleótidos de Adenina/metabolismo , Gránulos Cromafines/metabolismo , Etenoadenosina Trifosfato/metabolismo , Nucleótidos de Adenina/aislamiento & purificación , Animales , Transporte Biológico , Cromatografía Líquida de Alta Presión , CinéticaRESUMEN
The enzymatic syntheses of ATP analogs, such as tubercidin 5'-triphosphate, formycin A 5'-triphosphate, and etheno-ATP, from their respective mono- and diphosphate are described. The reaction products were purified by reverse-phase HPLC using a C-18 matrix and a volatile mobile phase at pH 7, with tributylamine as the ion-pairing agent. Each of the analogs required a buffer of somewhat different composition for the baseline separation of reaction product and reactants. The elutions were isocratic and allowed several successive runs without any intermediate equilibration of the column. After freeze-drying of the pooled fractions, the yield of the synthesized nucleoside triphosphate was approximately 70%. The described procedures are applicable either for analytical investigations or for semi-preparative purposes.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/síntesis química , Nucleótidos de Adenina/síntesis química , Nucleótidos de Adenina/aislamiento & purificación , Adenosina Difosfato/análisis , Adenosina Monofosfato/análisis , Adenosina Trifosfato/análisis , Adenosina Trifosfato/aislamiento & purificación , Adenilato Quinasa/metabolismo , Cromatografía Líquida de Alta Presión , Etenoadenosina Trifosfato/síntesis química , Etenoadenosina Trifosfato/aislamiento & purificación , Formicinas/síntesis química , Formicinas/aislamiento & purificación , Nucleósido-Fosfato Quinasa/metabolismo , Piruvato Quinasa/metabolismo , Ribonucleótidos/síntesis química , Ribonucleótidos/aislamiento & purificación , Tubercidina/análogos & derivados , Tubercidina/síntesis química , Tubercidina/aislamiento & purificaciónRESUMEN
We prepared nucleotide-free actin in buffer containing 48% (w/v) sucrose. Sucrose inhibits the irreversible denaturation of actin that follows nucleotide dissociation [Kasai et al. (1965) Biochim. Biophys. Acta 94, 494-503]. Our conditions removed nucleotide from approximately 80% of the actin. Stabilization of nucleotide-free actin depends on the sucrose concentration. The CD ellipticity (x 10(3) deg cm2 dmol-1) at 222 nm of nucleotide-free actin in 48% sucrose is -3.54. The ellipticity of denatured nucleotide-free actin in dilute buffer is -2.01 and that of native actin is -4.19. In 48% sucrose nucleotide-free actin has 1.12 and native actin has 0.5 solvent-exposed thiol residues. The conformation of native actin is recovered when ATP and Mg2+ are added. Our ability to generate stable nucleotide-free actin permitted us to study the kinetics of nucleotide binding to actin. The observed rate constant of the reaction is linearly dependent on the concentration of epsilon ATP, a fluorescent analog of ATP. The inverse of the association rate constant is proportional to the viscosity of the solvent with an intercept near the origin as expected for a diffusion-limited reaction. The second-order association rate constant for Mg(2+)-ATP and Ca(2+)-ATP binding to nucleotide-free actin in water at 22 degrees C is 5 x 10(6) M-1 s-1. The Smoluchowski collision rate constant for actin and ATP is calculated to be 6.5 x 10(9) M-1 s-1, which makes the "orientation factor" 7.7 x 10(-4). From the ratio of the dissociation and association rate constants, we calculate dissociation equilibrium constants of 1.2 x 10(-9) M for Mg(2+)-ATP-actin, 4.4 x 10(-9) M for Mg(2+)-epsilon ATP-actin, and 1.2 x 10(-10) M for Ca(2+)-ATP-actin.