RESUMEN
A new radioiodinated (2.2 Ci/mumol) iodocyanopindolol derivative carrying a 4-(3-trifluoromethyldiazirino)benzoyl residue has been synthesized. The long-wavelength absorption of the diazirine permits formation of the carbene by photolysis under very mild conditions. [125I]ICYP-diazirine binds with high affinity (Kd = 60 pM) to beta-receptors from turkey erythrocyte membranes. Upon irradiation, [125I]ICYP-diazirine is covalently incorporated in a Mr 40 000 protein. Stereoselective inhibition of photolabeling by the (-)enantiomers of alprenolol and isoproterenol indicated that the Mr 40 000 protein contains a beta-adrenergic binding site. The yield of specific labeling was up to 8.2% of total beta-receptor binding sites. The Mr 40 000 protein photolabeled in the membrane could be solubilized at comparable yield with either digitonin or Triton X-100. Irradiation of digitonin-solubilized turkey erythrocyte membranes with [125I]ICYP-diazirine resulted in specific labeling of two proteins with Mr 40 000 and 50 000. In guinea-pig lung membranes, at least five proteins were photolabeled, of which one (with approximately Mr 67 000) was labeled specifically.
Asunto(s)
Marcadores de Afinidad/metabolismo , Azirinas/metabolismo , Receptores Adrenérgicos beta/análisis , Receptores Adrenérgicos/análisis , Animales , Digitonina , Membrana Eritrocítica/metabolismo , Cobayas , Pulmón/metabolismo , Proteínas de la Membrana/metabolismo , Fotólisis , Receptores Adrenérgicos beta/metabolismo , Solubilidad , PavosRESUMEN
When monolayer cultured hepatocytes were incubated with 1 nM [125I]glucagon at 30 degrees C, equilibrium was reached after 10 min, whereas at 4 degrees C, equilibrium was reached after 60 min. At the higher temperature, 11.2% of the bound ligand was broken down after 60 min, at the lower temperature, the amount of degradation was negligible. At 30 degrees C, acid-washing did not remove specifically bound ligand; thus, it was assumed that the ligand was internalised at this temperature, since some of the specifically bound ligand could be washed off at lower temperatures. This was confirmed in experiments when monolayer cultures of hepatocytes were incubated with fluorescein-labelled derivatives of glucagon. The distribution of specific binding on the cell surface was studied at both 30 and 4 degrees C using video intensification microscopic techniques. In keeping with studies using radiolabelled glucagon, more fluorescence was detected following incubation at 4 degrees C than at 30 degrees C and it could be removed by washing the cells. Video intensification microscopy indicated that at the lower temperature, the bound ligand was distributed all over the cell surface. At the higher temperature, ligand-derived fluorescence could only be detected in mobile intracellular vesicles.
Asunto(s)
Membrana Celular/metabolismo , Colorantes Fluorescentes/metabolismo , Glucagón/análogos & derivados , Glucagón/metabolismo , Hígado/metabolismo , Receptores de la Hormona Gastrointestinal/metabolismo , Animales , Células Cultivadas , Cinética , Hígado/citología , Ratas , Receptores de GlucagónRESUMEN
The synthesis of monofluorescein, monorhodamine, and mono-4-nitrobenz-2-oxa-1,3-diazole (NBD) derivatives of glucagon is reported. The fluorescent groups were introduced by converting tryptophan-25 to 2-thioltryptophan using thiol-specific fluorescent reagents. All derivatives retained the ability to activate adenylate cyclase when compared to glucagon and thus were considered full agonists. IC50 values of 6.8.10(-9), 1.7.10(-8), 1.8.10(-8) and 5.4.10(-9) M were measured in rat liver membranes for NBD-, fluorescein-, rhodamine-Trp25-glucagon and native glucagon, respectively. From the IC50 values Kd values of 2.16.10(-9), 4.10(-9), 2.10(-9) and 1.72.10(-9) M were calculated for the binding of NBD-, fluorescein-, rhodamine-Trp25-glucagon and native glucagon, respectively. The highest quantum yield (0.18) of the monomer derivatives was obtained with fluorescein-Trp25-glucagon in phosphate-buffered saline (pH 7.4). Difluorescein-glucagon was also prepared by reacting the amino groups of histidine-1 and lysine-12 with fluorescein isothiocyanate and dimer derivatives were prepared using fluorescein-labelled 2-thiolTrp25-glucagon. Difluorescein-glucagon bound only weakly to glucagon receptors and displayed antagonist properties. The dimer derivative formed from two difluorescein-2-thiolTrp25-glucagon molecules had similar poor binding qualities, whereas the dimer formed from difluorescein-2-thiolTrp25-glucagon and 2-thiolTrp25-glucagon exhibited, at low concentrations, properties similar to monofluorescein-glucagon. Both dimer derivatives were only sparingly soluble in aqueous medium. Specific binding of fluorescein-Trp25-glucagon and difluorescein-glucagon to rat hepatocytes was followed using flow cytometry.
Asunto(s)
Colorantes Fluorescentes/síntesis química , Glucagón/análogos & derivados , Glucagón/síntesis química , Adenilil Ciclasas/metabolismo , Animales , Unión Competitiva , Membrana Celular/metabolismo , Células Cultivadas , Colorantes Fluorescentes/farmacología , Glucagón/metabolismo , Glucagón/farmacología , Indicadores y Reactivos , Cinética , Hígado/metabolismo , Ratas , Receptores de la Hormona Gastrointestinal/efectos de los fármacos , Receptores de la Hormona Gastrointestinal/metabolismo , Receptores de Glucagón , Relación Estructura-ActividadRESUMEN
The role of the beta gamma-subunits in the interaction of G-proteins was examined with beta 1-adrenoceptors purified from turkey erythrocytes and pure beta gamma-subunits prepared from turkey erythrocytes and bovine brain. On a non-denaturing polyacrylamide gel, the mobility of beta gamma-subunits was increased when incubated with beta 1-adrenoceptor and the beta 1-adrenergic agonist 1-(-)-isoproterenol, whereas on incubation with the antagonist 1-alprenolol the mobility was unchanged. Furthermore, the beta 1-adrenoceptor was retarded on a Sephadex G-50 column equilibrated with beta gamma-subunits and agonist. No retardation occurred in the presence of antagonist. These data suggest a direct interaction of activated beta 1-adrenoceptors with isolated beta gamma-subunits of G-proteins.
Asunto(s)
Encéfalo/metabolismo , Membrana Eritrocítica/metabolismo , Proteínas de Unión al GTP/metabolismo , Receptores Adrenérgicos beta/metabolismo , Animales , Bovinos , Electroforesis en Gel de Poliacrilamida , Sustancias Macromoleculares , Receptores Adrenérgicos beta/aislamiento & purificación , PavosRESUMEN
A human cDNA fragment bearing the complete coding region for the beta 2-adrenergic receptor was introduced into the genome of Autographa california nuclear polyhedrosis virus under the control of the polyhedrin promoter. Binding studies using [125I]iodocyanopindolol showed that Sf9 insect cells infected with the recombinant virus expressed approximately 1 x 10(6) beta 2-adrenergic receptors on their cell surface. Photoaffinity labeling of whole cells and membranes revealed a molecular weight of approximately 46,000 for the expressed receptor. The receptor produced in insect cells is glycosylated but the extent and pattern differ from that of the receptor from human tissue. The heterologously expressed receptor was purified by alprenolol affinity chromatography, and was able to activate isolated Gs-protein.
Asunto(s)
Receptores Adrenérgicos beta/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Adenilil Ciclasas/metabolismo , Alprenolol/metabolismo , Baculoviridae , Clonación Molecular , Proteínas de Unión al GTP/metabolismo , Vectores Genéticos , Glicosilación , Humanos , Técnicas In Vitro , Sustancias Macromoleculares , Receptores Adrenérgicos beta/química , Receptores Adrenérgicos beta/metabolismoRESUMEN
The regulation of adenylate cyclase by hormones and by GTP regulatory proteins was investigated in native membrane systems and in systems reconstituted from purified components. These studies can be summarized as follows. The stimulatory beta 1-adrenoceptor catalyses the activation of a complex between the GTP stimulatory protein GS and the catalytic unit C. The agonist-receptor complex can activate a few cyclase units in native membrane systems as well as in reconstituted systems. GS from turkey erythrocytes is functionally different from rabbit liver GS, the latter being more amenable to activation by guanyl nucleotides in the absence of hormone. The coupling between the beta 1-adrenoceptor GS and C is efficient when compared with the coupling obtained in native membrane systems. GTP/GDP exchange at the alpha S subunit requires the presence of the beta gamma subunits. A mechanism for the inhibition of adenylate cyclase by the inhibitory GTP regulatory protein Gi is suggested.
Asunto(s)
Adenilil Ciclasas/metabolismo , Animales , Fenómenos Químicos , Química , Eritrocitos/enzimología , Proteínas de Unión al GTP/fisiología , Técnicas In Vitro , Cinética , Receptores Adrenérgicos beta/fisiología , PavosRESUMEN
The synthesis and properties of a fluorescent derivative of the hydrophilic beta-adrenergic antagonist CGP-12177 are described. The fluorescence of the NBD derivative of CGP-12177 (CGP-NBD) is extremely sensitive to its environment, the quantum yield increasing 23-fold upon transfer from water to acetonitrile. This property of CGP-NBD was taken into account and a procedure was developed using quantitative chloroform extraction of ligand for the measurement of CGP-NBD bound specifically to beta-receptors on A431.E3 membranes. The fluorescent NBD-derivative of CGP-12177 bound strongly and specifically to A431 cells, a KD of 3.9 x 10(-10) M being measured; the specific binding represented 63% of the total binding at a concentration of 1 x 10(-8) M (256 x KD). A431.E3 cells were used for the binding studies since they gave consistently higher receptor numbers when compared with the native strain. A maximal number of 47,000 sites/cell and a KD of 100 pM were measured with CGP-12177 on adhered cells. The receptor number was strongly dependent upon cell density with only 3000 sites/cell being measured in suspension at confluence.
Asunto(s)
4-Cloro-7-nitrobenzofurazano/metabolismo , Oxadiazoles/metabolismo , Propanolaminas/metabolismo , Receptores Adrenérgicos beta/metabolismo , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/síntesis química , Antagonistas Adrenérgicos beta , Animales , Línea Celular , Células Cultivadas , Membrana Eritrocítica/metabolismo , Colorantes Fluorescentes , Cinética , Propanolaminas/síntesis química , Solventes , Espectrometría de Fluorescencia , PavosRESUMEN
A mechanism for the phosphorylase reaction is proposed which offers a plausible explanation for the essential role of pyridoxal 5'-phosphate in glycogen phosphorylases: in the forward direction, phosphorolysis of alpha-1,4-glycosidic bonds in oligo- or polysaccharides is started by protonation of the glycosidic oxygen by the substrate orthophosphate followed by stabilization of the incipient oxocarbonium ion and subsequent covalent binding to form alpha-glucose 1-phosphate. In the reverse direction, protonation of the phosphate of glucose 1-phosphate destabilizes the glycosidic bond and promotes formation of a glucosyl oxocarbonium ion-phosphate anion pair. In the subsequent step the phosphate anion facilitates the nucleophilic attack of a terminal glucosyl residue on the carbonium ion bringing about alpha-1,4-glycosidic bond formation and primer elongation. Both in the forward and reverse reactions, the phosphate of the cofactor pyridoxal 5'-phosphate acts as a general acid (PL-OPO3H- or PL-OPO3(2-) and protonates the substrate phosphate functioning as proton shuttle. Thus in glycogen phosphorylases, phosphates which directly interact with each other have replaced a pair of amino acid carboxyl groups functioning in catalysis of carbohydrases.
Asunto(s)
Fosforilasas/metabolismo , Fosfato de Piridoxal/metabolismo , Secuencia de Aminoácidos , Animales , Catálisis , Datos de Secuencia MolecularAsunto(s)
Proteínas de Unión al GTP/fisiología , Proteínas/química , Proteínas/fisiología , Transducción de Señal , Secuencia de Aminoácidos , Proteínas de Unión al GTP/química , Hormonas/química , Hormonas/fisiología , Proteínas de la Membrana/química , Proteínas de la Membrana/fisiología , Datos de Secuencia Molecular , Conformación Proteica , Receptores de Superficie Celular/química , Receptores de Superficie Celular/fisiología , Rodopsina/química , Rodopsina/fisiología , Relación Estructura-ActividadAsunto(s)
Bioquímica/historia , Biofisica/historia , Enzimas/historia , Alemania , Historia del Siglo XX , MissouriAsunto(s)
Fosforilasas/metabolismo , Fosfato de Piridoxal/metabolismo , Regulación Alostérica , Animales , Sitios de Unión , Catálisis , Fenómenos Químicos , Química , Escherichia coli/enzimología , Espectroscopía de Resonancia Magnética , Músculos/enzimología , Fosfatos/metabolismo , Plantas/enzimología , Unión Proteica , ConejosAsunto(s)
Adenilil Ciclasas/metabolismo , Membrana Eritrocítica/enzimología , Eritrocitos/enzimología , Nucleótidos de Guanina/metabolismo , Receptores de Superficie Celular , Adenilil Ciclasas/biosíntesis , Animales , Sitios de Unión , Columbidae , Activación Enzimática , Membrana Eritrocítica/metabolismo , Nucleótidos de Guanina/farmacología , Guanosina Trifosfato/metabolismo , Técnicas In Vitro , Isoproterenol/farmacología , Tubulina (Proteína)/metabolismoAsunto(s)
Adenilil Ciclasas/metabolismo , Hormonas/fisiología , Receptores Adrenérgicos beta/metabolismo , Receptores Adrenérgicos/metabolismo , Animales , Toxina del Cólera/farmacología , Columbidae , Dihidroalprenolol/metabolismo , Activación Enzimática , Membrana Eritrocítica/metabolismo , Guanosina Trifosfato/metabolismo , Unión ProteicaAsunto(s)
Biología Molecular/historia , Biología/historia , Historia del Siglo XX , Humanos , Ciencia/historiaRESUMEN
Published data revealing discrepancies between the kinetics of hormone binding and adenylate cyclase activation are critically analyzed. This analysis gave rise to the hypothesis concerning the translation of hormone receptor interactions into biological function, i.e.: adenylate cyclase activation. Because the lifetime of the hormone receptor complex is too long, other steps in signal transfer are considered for the control of adenylate cyclase activity.
Asunto(s)
Adenilil Ciclasas/metabolismo , Hormonas/metabolismo , Receptores de Superficie Celular , Animales , Sitios de Unión , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Columbidae , Activación Enzimática , Nucleótidos de Guanina/metabolismo , Humanos , Cinética , Ratones , Modelos Químicos , Unión Proteica , RatasRESUMEN
Information available at present documents the existence of three well-defined classes of guanine nucleotide binding proteins functioning as signal transducers: Gs and Gi which stimulate and inhibit adenylate cyclase, respectively, and transducin which transmits and amplifies the signal from light-activated rhodopsin to cGMP-dependent phosphodiesterase in ROS membranes. Go is a fourth member of this family. Its function is the least known among GTP binding signal transducing proteins. The family of G proteins has a number of properties in common. All are heterotrimers consisting of three subunits, alpha, beta, and gamma. Each of the subunits may be heterogeneous depending on species and tissue of origin and may be posttranslationally modified covalently. The alpha subunits vary in size from 39 to 52 kDa. The sequences for Gs alpha and transducin alpha have 42% overall homology and those of Gi alpha and Gs alpha 43%, whereas those of Gi alpha and transducin alpha have a higher degree (68%) of homology. All alpha subunits bind guanine nucleotides and are ADP-ribosylated by either pertussis toxin (Gi, transducin, Go) or cholera toxin (Gs, Gi, transducin). Thus, transducin and Gi, which have the highest degree of sequence homology, are also ADP-ribosylated by both toxins. The beta subunits have molecular weights of 36 and 35 kDa, respectively. While Gs, Gi, and Go contain a mixture of both, transducin contains only the larger (36-kDa) beta-polypeptide. The relationship of the 36- and the 35-kDa beta subunits is not defined. Although the complete sequence of the 36-kDa beta subunit of transducin has been deduced from the cDNA sequence, complete sequences of other beta subunits are not yet available so that detailed comparisons cannot be made at present. However, the proteolytic profiles of each class of the beta subunits of different G proteins are indistinguishable. The gamma subunit of bovine transducin has been completely sequenced. It has a Mr of 8400. Again complete sequences of other gamma subunits are not yet available. While the gamma subunits of Gs, Gi, and Go have identical electrophoretic mobility in SDS gels, they differ significantly in this respect from the gamma subunit of transducin. Moreover, crossover experiments point to functional differences between gamma subunits from G protein and transducin complexes. In addition, a role for beta, gamma in anchoring guanine nucleotide binding proteins to membranes has been postulated.(ABSTRACT TRUNCATED AT 400 WORDS)