RESUMEN
Familial amyloidotic polyneuropathy (FAP) is linked to destabilising point mutations in the human plasma protein transthyretin (TTR). Consistent with similar amyloid disorders, low molecular weight TTR oligomers have been shown to exert the major cytotoxic effect. The amyloid structure of TTR contains non-native inter-molecular disulphide linkages via the cysteine at position 10 (Cys10). Moreover, substitution of Cys10 in a mouse model for TTR-amyloidosis abolishes TTR deposits, indicating an important role of Cys10 in FAP pathogenesis. However, the role of disulphide bridges in TTR cytotoxicity has not been elucidated. By probing Cys10Ser TTR variants to the human neuroblastoma SH-SY5Y cell line, we have addressed this question, and our results clearly show that formation of an inter-molecular disulphide bridge is not a pre-requisite for TTR cytotoxicity. This finding suggests that prevention of inter-molecular TTR disulphide bridges as a therapeutic intervention will not impair the cytotoxic potential of TTR.
Asunto(s)
Amiloide/química , Prealbúmina/química , Sustitución de Aminoácidos , Amiloide/toxicidad , Neuropatías Amiloides Familiares/etiología , Neuropatías Amiloides Familiares/genética , Neuropatías Amiloides Familiares/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cisteína/química , Disulfuros/química , Humanos , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/toxicidad , Mutagénesis Sitio-Dirigida , Prealbúmina/genética , Prealbúmina/toxicidad , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/toxicidadRESUMEN
The majority of early-onset familial Alzheimer disease cases are caused by mutations in the genes encoding presenilin 1 (PS1) and presenilin 2 (PS2). Presenilin mutations have been hypothesised to cause Alzheimer disease either by altering amyloid precursor protein metabolism or by increasing the vulnerability of neurons to undergo death by apoptosis. We showed previously that PS1 exon 9 deletion (PS1 DeltaE9) and L250S mutations predispose SH-SY5Y neuroblastoma cells to high glucose stress-induced apoptosis and that the anti-apoptotic effect of insulin-like growth factor I (IGF-I) is compromised by these mutations. The present study investigates whether the susceptibility of PS1 mutation transfected SH-SY5Y cells to undergo apoptosis is likely due to a downregulation of Akt/protein kinase B (Akt), a key intermediate in the phosphatidylinositol 3 (PI3)-kinase arm of the IGF-I signaling pathway. We used two methods to determine the regulation of Akt in response to the pro-apoptotic stimuli of serum deprivation and high glucose stress, as well as treatment with IGF-I. We also looked at the phosphorylatiom state of GSK-3beta at Ser9. Using a kinase assay with immunoprecipitated Akt, we detected an increased Akt activity in PS1 L250S cells at 1 hr after the combination of 20 mM glucose plus 10 nM IGF-I, when compared to the other cell types. This effect, however, was transient in that no mutation related differences were seen at either 6- or 24-hr post-treatment. Immunoblotting for Phospho-Akt as a ratio of total Akt, as well as for GSK-3beta phosphorylated at Ser9 revealed no apparent between cell type and treatment differences. This data strongly indicates that PS1 wt and mutant cells show no major differences in the pattern of Akt regulation after exposure to the pro-apoptotic stimuli of either serum deprivation or high glucose stress, or treatment with IGF-I. It is suggested that another component of IGF-I signaling is likely disrupted in these cells to increase their vulnerability to undergo death by apoptosis.
Asunto(s)
Apoptosis/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Proteínas de la Membrana/genética , Mutación/fisiología , Neuronas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Estrés Fisiológico/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/fisiopatología , Apoptosis/efectos de los fármacos , Proteínas Sanguíneas/deficiencia , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Glucosa/farmacología , Glucógeno Sintasa Quinasa 3 , Humanos , Immunoblotting , Factor I del Crecimiento Similar a la Insulina/farmacología , Proteínas de la Membrana/metabolismo , Neuroblastoma , Fosforilación/efectos de los fármacos , Presenilina-1 , Proteínas Proto-Oncogénicas c-akt , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Estrés Fisiológico/fisiopatología , TransfecciónRESUMEN
Presenilin 1 (PS1) regulates beta-catenin stability; however, published data regarding the direction of the effect are contradictory. We examined the effects of wild-type and mutant forms of PS1 on the membrane, cytoplasmic, nuclear, and signaling pools of endogenous and exogenous beta-catenin by immunofluorescence microscopy, subcellular fractionation, and in a transcription assay. We found that PS1 destabilizes the cytoplasmic and nuclear pools of beta-catenin when stabilized by Wnt or Dvl but not when stabilized at lower levels of the Wnt pathway. The PS1 mutants examined were less able to reduce the stability of beta-catenin. PS1 also inhibited the transcriptional activity of endogenous beta-catenin, and the PS1 mutants were again less inhibitory at the level of Dvl but showed a different pattern of inhibition toward transcription below Dvl. The transcriptional activity of exogenously expressed wild-type beta-catenin and two mutants, DeltaN89beta-catenin and DeltaSTbeta-catenin, were also inhibited by wild-type and mutant PS1. We conclude that PS1 negatively regulates the stability and transcriptional activity of beta-catenin at different levels in the Wnt pathway, that the effect on transcriptional activity appears to be independent of the GSK-3beta mediated degradation of beta-catenin, and that mutations in PS1 differentially affect the stability and transcriptional activity of beta-catenin.
Asunto(s)
Proteínas Portadoras , Proteínas del Citoesqueleto/metabolismo , Proteínas de la Membrana/fisiología , Proteínas de Neoplasias , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores , Transcripción Genética , Proteínas de Pez Cebra , Proteínas Adaptadoras Transductoras de Señales , Animales , Western Blotting , Línea Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Litio/farmacología , Luciferasas/genética , Microscopía Fluorescente , Presenilina-1 , Transducción de Señal , Fracciones Subcelulares/metabolismo , Proteínas Wnt , beta CateninaRESUMEN
The single-turnover kinetic mechanism for the reaction catalyzed by dTDP-glucose 4,6-dehydratase (4,6-dehydratase) has been determined by rapid mix-chemical quench mass spectrometry. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was employed to analyze quenched samples. The results were compatible with the postulated reaction mechanism, in which NAD(+) initially oxidizes glucosyl C4 of dTDP-glucose to NADH and dTDP-4-ketoglucose. Next, water is eliminated between C5 and C6 of dTDP-4-ketoglucose to form dTDP-4-ketoglucose-5,6-ene. Hydride transfer from NADH to C6 of dTDP-4-ketoglucose-5,6-ene regenerates NAD(+) and produces the product dTDP-4-keto-6-deoxyglucose. The single-turnover reaction was quenched at various times on the millisecond scale with a mixture of 6 M guanidine hydrochloride and sodium borohydride, which stopped the reaction and reductively stabilized the intermediates and product. Quantitative MALDI-TOF MS analysis of the quenched samples allowed the simultaneous observation of the disappearance of substrate, transient appearance and disappearance of dTDP-hexopyranose-5,6-ene (the reductively stabilized dTDP-4-ketoglucose-5,6-ene), and the appearance of product. Kinetic modeling of the process allowed rate constants for most of the steps of the reaction of dTDP-glucose-d(7) to be evaluated. The transient formation and reaction of dTDP-4-ketoglucose could not be observed, because this intermediate did not accumulate to detectable concentrations.
Asunto(s)
Glucosa/análogos & derivados , Hidroliasas/química , Borohidruros/química , Catálisis , Deuterio/química , Estabilidad de Enzimas , Escherichia coli/enzimología , Glucosa/química , Cinética , Oxidación-Reducción , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrofotometría/métodos , Relación Estructura-Actividad , Especificidad por Sustrato , Nucleótidos de Timina/químicaRESUMEN
Non-amyloidogenic alpha-secretase processing of amyloid precursor protein (APP) is stimulated by protein kinase C (PKC). Levels and activity of PKC are decreased in sporadic Alzheimer's disease skin fibroblasts. We investigated whether alterations in PKC and PKC-mediated APP processing occur also in fibroblasts established from individuals with familial Alzheimer's disease APP KM670/671NL, PS1 M146V and H163Y mutations. These pathogenic mutations are known to alter APP metabolism to increase Abeta. PKC activities, but not levels, were decreased by 50% in soluble fractions from sporadic Alzheimer's disease cases. In contrast, familial Alzheimer's disease fibroblasts showed no significant changes in PKC enzyme activity. Fibroblasts bearing the APP KM670/671NL mutation showed no significant differences in either PKC levels or PKC-mediated soluble APP (APPs) secretion, compared to controls. Fibroblasts bearing PS1 M146V and H163Y mutations showed a 30% increase in soluble PKC levels and a 40% decrease in PKC-mediated APPs secretion. These results indicate that PKC deficits are unlikely to contribute to increased Abeta seen with APP and PS1 mutations, and also that PS1 mutations decrease alpha-secretase derived APPs production independently of altered PKC activity.
Asunto(s)
Enfermedad de Alzheimer/enzimología , Endopeptidasas/metabolismo , Proteína Quinasa C/metabolismo , Piel/enzimología , Adulto , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/genética , Secretasas de la Proteína Precursora del Amiloide , Ácido Aspártico Endopeptidasas , Células Cultivadas , Medios de Cultivo/química , Endopeptidasas/análisis , Endopeptidasas/genética , Femenino , Fibroblastos/enzimología , Humanos , Masculino , Persona de Mediana Edad , Mutación , Proteína Quinasa C/análisis , SueciaRESUMEN
Alzheimer's disease (AD) is associated with a striking reduction in the activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC). The deficiency occurs in brains from AD patients of undefined etiology, and in fibroblasts from both sporadic and familial AD cases. To further assess the nature of the abnormality of KGDHC in AD, KGDHC activities and immunoreactivities were analyzed in brains from AD patients bearing the Swedish APP670/671 mutation. This gene defect causes overproduction of the amyloid beta peptide. KGDHC activities were reduced by 55 to 57% compared with control values in the mutation-bearing AD cases in the medial temporal and superior frontal cortices. The immunochemical levels of KGDHC subunits Elk (-51%) and E2k (-76%) declined, whereas E3 concentrations were unchanged. The results suggest that mitochondrial dysfunction is a part of the pathophysiological process in AD even when the primary pathogenic cause is nonmitochondrial.
Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Aciltransferasas/metabolismo , Anciano , Glutamato Deshidrogenasa/metabolismo , Humanos , Persona de Mediana Edad , Distribución TisularRESUMEN
Electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry allows for high-resolution, accurate mass analysis of multiply charged ions of proteins. In the work described here, the ability of ESI-FTICR to distinguish small differences in molecular mass is evaluated. Ubiquitin was used as an internal mass calibration standard to measure the molecular mass of cytochrome c, myoglobin, and several carbonic anhydrase isoforms. Mass calibration was based on the tallest isotopic peak of each ubiquitin charge state. Ubiquitin performed well as an internal standard because its charge states covered the appropriate mass range, interference was minimal, and the tallest peak was easily identified. The peak masses of cytochrome c (12.5 kDa) and myoglobin (17 kDa) were measured to an accuracy of about 0.02 Da (<2ppm). However, errors of 1.0 Da were observed for some individual determinations because of the difficulty in identifying the tallest peak. When the technique was applied to bovine carbonic anhydrase II, even combining data from several charge states did not yield an unequivocal assignment of the tallest peak, resulting in a mass assignment of 29,023.7 or 29,024.7. Similarly, measurements of two isoforms with a mass difference of 1 Da, human carbonic anhydrase I, pI 6.0 and 6.6, yielded overlapping values for the mass of the tallest peak. However, these two isoforms were clearly distinguished by (a) identification of the tallest peak using a measurement of average mass as a guide and (b) comparison of the isotopic peak intensity patterns.
Asunto(s)
Apoproteínas/química , Anhidrasas Carbónicas/química , Grupo Citocromo c/química , Mioglobina/química , Proteínas/química , Animales , Calibración , Bovinos , Cromatografía Líquida de Alta Presión/métodos , Ciclotrones , Análisis de Fourier , Humanos , Isoenzimas/química , Espectrometría de Masas/instrumentación , Espectrometría de Masas/métodos , Peso Molecular , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Several mutations causing early-onset familial Alzheimer's disease (AD) have been detected in the presenilin 1 (PS-1) gene. Pathogenic mutations have also been described in an homologous gene, presenilin 2 (PS-2). In order to screen for mutations in these genes, cDNA samples from early-onset AD cases were analysed, using single strand conformation polymorphism (SSCP) and direct cDNA sequencing. Two missense mutations in the PS-1 gene were detected, a previously unidentified amino acid substitution Leu262Phe and an earlier reported amino acid substitution Glu318Gly. No disease-related mutations were found in the PS-2 gene.
Asunto(s)
Enfermedad de Alzheimer/genética , Sustitución de Aminoácidos , Leucina , Proteínas de la Membrana/genética , Fenilalanina , Mutación Puntual , Edad de Inicio , Pruebas Genéticas/métodos , Humanos , Polimorfismo Conformacional Retorcido-Simple , Presenilina-1 , Presenilina-2 , Análisis de Secuencia de ADNRESUMEN
beta-Adrenoceptor- and forskolin-stimulated adenylyl cyclase activities were determined in primary skin fibroblasts established from patients with sporadic Alzheimer's disease (AD) and from individuals with familial APP KM670/671NL, PS1 M146V and PS1 H163Y mutations. Our data showed a significantly decreased beta-adrenoceptor-stimulated adenylyl cyclase activity in fibroblasts from sporadic AD compared with age-matched controls (p < 0.001, Student's unpaired t-test). In contrast, both beta-adrenoceptor- and forskolin-stimulated adenylyl cyclase activities were significantly increased in fibroblasts bearing PS1 M146V and PS1 H163Y mutations compared with controls (p < 0.01 and p < 0.05, respectively). No differences were seen between cell lines with and without the Swedish APP KM670/671NL double mutation. We suggest that various gene mutations associated with AD have different consequences for the regulation of adenylyl cyclase signal transduction in this disorder.
Asunto(s)
Adenilil Ciclasas/metabolismo , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Regulación Enzimológica de la Expresión Génica/fisiología , Proteínas de la Membrana/genética , Agonistas Adrenérgicos beta/farmacología , Adulto , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/biosíntesis , Línea Celular , Colforsina/farmacología , Femenino , Fibroblastos/enzimología , Humanos , Isoproterenol/farmacología , Masculino , Proteínas de la Membrana/biosíntesis , Persona de Mediana Edad , Mutación/fisiología , Presenilina-1 , Transducción de Señal/fisiología , Piel/citologíaRESUMEN
Abnormalities in cultured fibroblasts from familial Alzheimer's Disease (FAD) cases uniquely enable the determination of how gene defects alter cell biology in living tissue from affected individuals. The current study focused on measures of calcium regulation and oxidative metabolism in fibroblast lines from controls and FAD individuals with the Swedish APP670/671 mutation. Bombesin-induced elevations in calcium in APP670/671 mutation-bearing lines were reduced by 40% (p < 0.05), a striking contrast to the 100% increase seen in sporadic AD and presenilin-1 (PS1) mutation-bearing cells in previously published studies. The APP670/671 mutation-bearing lines did not exhibit the exaggerated 4-bromo-A23187 releasable pool of calcium following 10 nM bradykinin, the enhanced sensitivity of calcium stores to low concentrations of bradykinin, nor the reduced activity of alpha-ketoglutarate dehydrogenase previously reported in cells from sporadic AD and mutant PS1 FAD. Thus, an altered regulation of internal calcium stores is common to all AD lines, but the calcium pool affected and the polarity of the alteration varies, apparently in association with particular gene mutations. Comparison of signal transduction in cell lines from multiple, genetically characterized AD families will allow testing of the hypothesis that these various pathogenic FAD abnormalities that lead to AD converge at the level of abnormal signal transduction.
Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/biosíntesis , Precursor de Proteína beta-Amiloide/genética , Mutación/fisiología , Enfermedad de Alzheimer/enzimología , Bombesina/farmacología , Bradiquinina/metabolismo , Calcimicina/farmacología , Calcio/metabolismo , Fibroblastos/enzimología , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Humanos , Ionóforos/farmacología , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Mitocondrias/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Derivatized bovine adenosine deaminase is used in enzyme replacement therapy and as an adjunct to gene therapy against severe combined immunodeficiency syndrome. Although a gene sequence is known for human adenosine deaminase, the structure of the bovine enzyme has not been characterized. Structure studies using mass spectrometry are reported here that evaluate sequence, processing, post-translational modifications and the extent of homology between the human protein and its therapeutic surrogate.
Asunto(s)
Adenosina Desaminasa/química , Adenosina Desaminasa/genética , Secuencia de Aminoácidos , Animales , Bovinos , Humanos , Espectrometría de Masas , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
Electroblotting proteins separated by gel electrophoresis provides a suitable support for further manipulations and analysis of small amounts of relatively pure samples. On-membrane digestion, peptide mapping by mass spectrometry, and database searching offer sensitive and fast tools to identify the analyte. By providing sequence information, tandem mass spectrometry can go a step further, confirming the database identification, solving problems connected with post-translational modifications and sequence variations, or supplying the stretches of internal sequence necessary to synthesize an oligonucleotide probe for gene isolation. The viability of this approach was successfully evaluated using different tandem mass spectrometric techniques: metastable decomposition in a matrix-assisted laser desorption/ionization (MALDI) time-of-flight instrument with a curved-field reflectron; low energy collision-induced dissociation in a MALDI quadrupole ion trap mass spectrometer; and high energy collision-induced dissociation in a high-performance four-sector mass spectrometer with massive cluster-impact ionization.
Asunto(s)
Proteínas/análisis , Secuencia de Aminoácidos , Electroforesis en Gel de Poliacrilamida , Hidrólisis , Datos de Secuencia Molecular , Péptidos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , TripsinaRESUMEN
We have characterised the muscarinic receptor subtypes found in human skin fibroblasts and compared binding levels in cell lines from members of the Alzheimer's disease family with the Swedish amyloid precursor protein (APP) 670/671 mutation. Binding studies with [3H] quinuclidinyl benzilate ([3H]QNB) and the M2/M4 selective antagonist [3H] (+/-)-5,11-dihydro-11-([(2-[(di-propylamino)methyl]-1- piperidinyl]ethyl)amino]carbonyl)-6H-pyrido(2,3-b)(1,4)benzodiazepine-6- one ([3H]AF-DX 384) revealed the presence of a single population of muscarinic receptors on lysed fibroblast membranes. [3H]QNB binding was displaced by a number of selective muscarinic ligands with a rank order of potency: atropine > himbacine > methoctramine > (+/-)-p-fluoro-hexahydro-sila-difenidol hydrochloride > pirenzepine > muscarinic-toxin-3. APP 670/671 mutation carrying cell lines showed 25-35% lower levels of muscarinic receptors labelled with [3H]QNB, [3H]N-methyl scopolamine and [3H]AF-DX 384, compared to controls. This difference was not statistically significant due to large individual variation. It is concluded that muscarinic receptors on adult skin fibroblasts are predominantly of the M2 subtype. Since these cells do not possess M1 and M3 receptor subtypes, they are unlikely to provide a good model for studying muscarinic receptor regulation of APP processing.
Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/genética , Mutación , Receptores Muscarínicos/metabolismo , Piel/química , Anciano , Células Cultivadas , Fibroblastos/química , Humanos , N-Metilescopolamina , Parasimpatolíticos/metabolismo , Pirenzepina/análogos & derivados , Pirenzepina/metabolismo , Quinuclidinil Bencilato/metabolismo , Valores de Referencia , Derivados de Escopolamina/metabolismo , Piel/patología , SueciaRESUMEN
Mass spectrometry has been used to define the reaction of BNPS-skatole (3-bromo-3-methyl-2-(o-nitrophenylsulfenyl)indolenine) with selected proteins and peptides, confirming the cleavage of the protein chain at tryptophan residues, characterizing the products, and identifying the side reactions. A short procedure for carrying out the cleavage reaction with minimal side reactions is presented.
Asunto(s)
Proteínas/química , Triptófano/química , Alquilación , Secuencia de Aminoácidos , Angiotensina I/química , Animales , Caballos , Indicadores y Reactivos , Espectrometría de Masas , Meliteno/química , Datos de Secuencia Molecular , Oxidación-Reducción , Escatol/análogos & derivados , Somatostatina/química , Somatostatina-28 , Triosa-Fosfato Isomerasa/químicaRESUMEN
Base treatment of polyethylene glycol-derivatized superoxide dismutase in which the polyethylene glycol is linked to the protein via a succinyl bridge, removes the polyethylene glycol leaving a succinyl marker. Exhaustive succinylation with d4-succinic anhydride completes the derivatization in order to minimize fractionation in proteolysis, chromatography, and desorption in the mass spectrometer. Production of peptides from the derivatized protein for high resolution and high-resolution tandem MS allows identification of the site that had been derivatized by polyethylene glycol and the determination of the amount of polyethylene glycol originally at each site. The mass spectrometric strategy outlined herein can be applied to other proteins derivatized for therapeutic administration.
Asunto(s)
Polietilenglicoles/análisis , Superóxido Dismutasa/análisis , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Endopeptidasas/metabolismo , Sustancias Macromoleculares , Espectrometría de Masas , Datos de Secuencia Molecular , Polietilenglicoles/metabolismo , Espectrometría de Masa Bombardeada por Átomos Veloces , Superóxido Dismutasa/metabolismoRESUMEN
The efficacy of beta 1-adrenoceptor-G protein coupling was studied in postmortem temporal cortex synaptic membranes from a series of control and Alzheimer's disease subjects. For the control cases, the non-hydrolysable GTP analogue 5'-guanylylimidodiphosphate (Gpp[NH]p) gave a significant reduction in the affinity of the agonist isoprenaline to displace binding of the radiolabelled antagonist (+/)-4-(3-t-butylamino-2-hydroxypropoxy)[5,7-3H]benzimidazol-2-one ([3H]CGP-12177). This effect was attributed to the conversion of high agonist-affinity sites to a lower-affinity state and was not found for the Alzheimer's disease cases. These data indicate that a disruption of beta 1-adrenoceptor-G protein coupling occurs in the temporal cortex of Alzheimer's disease patients.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Proteínas de Unión al GTP/metabolismo , Receptores Adrenérgicos beta/metabolismo , Membranas Sinápticas/metabolismo , Lóbulo Temporal/metabolismo , Anciano , Unión Competitiva , Femenino , Guanilil Imidodifosfato/farmacología , Humanos , Isoproterenol/metabolismo , Isoproterenol/farmacología , Masculino , Persona de Mediana Edad , Propanolaminas/metabolismo , Propanolaminas/farmacología , Unión Proteica/efectos de los fármacos , Receptores Adrenérgicos beta/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
The past year has seen greatly increased acceptance and application of the analytical capabilities of mass spectrometry by the biochemical community. The technique has been used to provide accurate mass determinations of non-covalently bound protein complexes, rapid mapping of molecular weights of altered peptides in protease digests, sequencing by collisional activation in tandem mass spectrometry, characterization of glycosylation and other modifications, and quantitation of peptides used in clinical diagnostics.
Asunto(s)
Espectrometría de Masas , Proteínas/química , Animales , HumanosRESUMEN
The value of the mass balance strategy is illustrated in the sequence determination of S. aureus V8 protease. Capillary electrophoresis, electrospray mass spectrometry, and high performance tandem mass spectrometry are used as well as proteolysis and Edman degradation. The carboxy terminus is found to contain 17 irregularly repeating units of the triptych motifs NNP and DNP, which provide a challenge to any strategy involving mapping, sequencing, and overlapping of hydrolytic peptides.
Asunto(s)
Proteínas/química , Análisis de Secuencia/métodos , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Bromuro de Cianógeno/química , Dinitrofenoles/análisis , Dinitrofenoles/química , Electroforesis/métodos , Espectrometría de Masas , Datos de Secuencia Molecular , Peso Molecular , Oligopéptidos/análisis , Oligopéptidos/química , Fragmentos de Péptidos/análisis , Mapeo Peptídico , Secuencias Repetitivas de Ácidos Nucleicos , Serina Endopeptidasas/químicaRESUMEN
Gamma-aminobutyric acid-B (GABAB) binding sites labelled with [3H]GABA were determined in postmortem frontal cortex samples of 20 control subjects and 16 suicides. The suicide group was further subdivided according to the method of suicide and the existence of depressive symptoms prior to death. No significant differences in GABAB binding were found either between overall suicide and control groups or between the control group and the other subgroups (violent suicide, nonviolent suicide, nondepressed and depressed suicide victims). A significant increase in GABAB binding was observed in those individuals dying from carbon monoxide poisoning. It is concluded that although GABAB binding sites are not altered in our suicide group, a presynaptic dysfunction might account for the increased GABAB binding found in the carbon monoxide subgroup.