RESUMEN
The catabolic enzyme allantoinase is rapidly inactivated in cells of Pseudomonas aeruginosa when the stationary phase of growth is reached. This process is irreversible since the protein synthesis inhibitor chloramphenicol completely blocked the reappearance of allantoinase activity that is observed when allantoin is added to stationary cells. Purified alloantoinase appeared to be a protein composed of four identical subunits with a molecular weight of 38,000. With antibodies raised against purified allantoinase it was found that allantoinase inactivation is accompanied by a parallel decrease in immunologically reactive material. This suggests that allantoinase inactivation is caused or followed by rapid proteolysis.
Asunto(s)
Alantoína/metabolismo , Amidohidrolasas/metabolismo , Pseudomonas aeruginosa/enzimología , Amidohidrolasas/aislamiento & purificación , Cloranfenicol/farmacología , Inmunodifusión , Cinética , Sustancias Macromoleculares , Peso MolecularRESUMEN
The 'high ammonia pathway' enzyme glutamate dehydrogenase (NADP+) is inactivated in cells of Pseudomonas aeruginosa when the stationary phase of growth is reached. Purified glutamate dehydrogenase (NADP+) appeared to be a protein composed of six identical subunits with a molecular weight of 54 000. With antibodies raised against purified enzyme it was found that glutamate dehydrogenase (NADP+) inactivation is accompanied by a parallel decrease in immunologically reactive material. This suggests that glutamate dehydrogenase (NADP+) inactivation is caused or followed by rapid proteolysis.
Asunto(s)
Glutamato Deshidrogenasa/aislamiento & purificación , Pseudomonas aeruginosa/enzimología , Complejo Antígeno-Anticuerpo , Reacciones Cruzadas , Glutamato Deshidrogenasa/antagonistas & inhibidores , Glutamato Deshidrogenasa/metabolismo , Sueros Inmunes , Inmunodifusión , Cinética , NADP/metabolismoRESUMEN
Subcellular fractions from SV-40 transformed hamster lens cells, prepared by chemical extractions, were tested for the presence of T-antigen by immunoautoradiography. Most of the T-antigen was present in the nucleus and was resistant to extraction by 2 M NaCl, indicating an association with the nuclear matrix. Another part of the T-antigen was, under certain conditions, resistant to extraction of the cells with a nonionic detergent. This T-antigen could be solubilized by Ca2+ at low temperature, conditions that also cause a specific depolymerization of microtubules.
Asunto(s)
Antígenos Virales de Tumores/genética , Transformación Celular Viral , Virus 40 de los Simios/inmunología , Animales , Antígenos Virales de Tumores/aislamiento & purificación , Núcleo Celular/inmunología , Células Cultivadas , Cricetinae , Técnica del Anticuerpo Fluorescente , Cristalino/fisiología , Peso Molecular , Fracciones Subcelulares/inmunologíaAsunto(s)
Acetilcolina/metabolismo , Duodeno/efectos de los fármacos , Agonistas de los Receptores Histamínicos/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Receptores Histamínicos/metabolismo , Transmisión Sináptica/efectos de los fármacos , Animales , Duodeno/fisiología , Guanidinas/farmacología , Humanos , Ligandos , Masculino , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Piperazinas/farmacología , Quinazolinonas/farmacología , Ratas , Ratas Wistar , Receptores Histamínicos H4 , Tiourea/análogos & derivados , Tiourea/farmacologíaRESUMEN
In the past years we developed high-resolution screening platforms involving separation of bioactive mixtures and on-line or at-line bioassays for a wide variety of biological targets with parallel mass spectrometric detection and identification. In the current research, we make a major step forward in the development of at-line bioassays by implementation of radioligand receptor binding and functional cellular assays to evaluate bioactvity and selectivity. We demonstrate a new platform for high-resolution metabolic profiling of lead compounds for functional activity and selectivity toward the human histamine H(4) receptor (hH(4)R), a member of the large family of membrane-bound G protein-coupled receptors. In this platform analytical chemistry, cell biology and pharmacology are merged. The methodology is based on chromatographic separation of metabolic mixtures by HPLC coupled to high-resolution fractionation onto (multiple) microtiter well plates for complementary assaying. The methodology was used for efficient and rapid metabolic profiling of the drug clozapine and three selective hH(4)R lead compounds. With this new platform metabolites with undesired alterations in target selectivity profiles can be readily identified. Moreover, the parallel identification of metabolite structures, with accurate-mass measurements and MS/MS, allowed identification of liable metabolic 'hotspots' for further lead optimization and plays a central role in the workflow and in this study. The methodology can be easily adapted for use with other receptor screening formats. The efficient combination of pharmacological assays with analytical techniques by leveraging high-resolution at-line fractionation as a linking technology will allow implementation of comprehensive metabolic profiling in an early phase of the drug discovery process.
Asunto(s)
Descubrimiento de Drogas/métodos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Histamínicos/metabolismo , Espectrometría de Masas en Tándem/métodos , Línea Celular Tumoral , Membrana Celular/metabolismo , Cromatografía Liquida , Clozapina/química , Clozapina/metabolismo , Células HEK293 , Histamina , Humanos , Ligandos , Piperazinas/química , Piperazinas/metabolismo , Quinazolinonas/química , Quinazolinonas/metabolismo , Ensayo de Unión Radioligante , Receptores Acoplados a Proteínas G/química , Receptores Histamínicos/química , Receptores Histamínicos H4Asunto(s)
Hígado/enzimología , Hidrolasas Diéster Fosfóricas/metabolismo , Animales , Sitios de Unión , Cromatografía en Gel , Cromatografía por Intercambio Iónico , AMP Cíclico , GMP Cíclico/farmacología , Estabilidad de Medicamentos , Activación Enzimática , Femenino , Calor , Imidazoles/farmacología , Cinética , Unión Proteica , Ratas , Dodecil Sulfato de Sodio/farmacología , Teofilina/farmacología , Factores de Tiempo , TritioRESUMEN
Both allantoinase and NADP-GDH in Pseudomonas aeruginosa were inactivated when cells reached the stationary phase of growth. Mutants unable to inactivate these enzymes were isolated. Results with recombinants showed that the mutation is not located in the structural genes of these enzymes but in an independent gene involved in the inactivation.