Asunto(s)
Humanos , Inmunidad Celular/fisiología , Biomarcadores/análisis , Pteridinas/inmunología , Infecciones Bacterianas/inmunología , Cromatografía Líquida de Alta Presión , Ensayo de Inmunoadsorción Enzimática/estadística & datos numéricos , Rechazo de Injerto/inmunología , Infecciones por VIH/inmunología , Trasplante de Riñón/inmunología , Neoplasias/inmunología , Pteridinas/sangre , Pteridinas/orina , Radioinmunodetección , Valores de Referencia , Virosis/inmunologíaRESUMEN
A monoclonal anti-idiotype antibody, NS7, previously shown to mimic the binding of the pterin cofactor of phenylalanine hydroxylase (phenylalanine 4-monooxygenase, EC 1.14.16.1) has been used to localize the cofactor binding site within the phenylalanine hydroxylase catalytic domain to a 27-amino-acid sequence that is highly conserved among the three aromatic amino acid hydroxylases. The binding of NS7 to a synthetic peptide corresponding to the phenylalanine hydroxylase sequence from residue 263 to residue 289 was blocked by the competitive inhibitor of phenylalanine hydroxylase enzyme activity, 7,8-dihydro-6,7-dimethylpterin. In addition this peptide competed with native phenylalanine hydroxylase for binding to 6,7-dimethyl-5,6,7,8-tetrahydropterin conjugated to a polyglutamate carrier. Application of this simple and direct approach to other enzymes is likely to greatly facilitate the identification of ligand binding sites on enzymes, which will significantly contribute to the understanding of enzyme structure-function relationships.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Idiotipos de Inmunoglobulinas/inmunología , Fenilalanina Hidroxilasa/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Sitios de Unión , Técnicas In Vitro , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fenilalanina Hidroxilasa/química , Pteridinas/inmunología , Pterinas/metabolismo , RatasRESUMEN
BALB/c mice were immunized with a synthetic co-factor of the aromatic amino acid hydroxylases, 6,7-dimethyl-5,6,7,8-tetrahydropterin, conjugated to albumin. Hybridoma cell lines isolated from the immunized mice secreted monoclonal antibodies reacting specifically with the pterin molecule and monoclonal antibodies which were found to bind phenylalanine hydroxylase. Several lines of evidence were consistent with the anti-phenylalanine hydroxylase antibodies being anti-idiotype antibodies mimicking the pterin molecule and binding to the pterin binding site of phenylalanine hydroxylase. (a) An anti-idiotype monoclonal antibody, NS7, when reimmunized into mice produced anti-pterin antibodies consistent with NS7 being an internal image anti-idiotypic antibody. (b) NS7 antibody was prevented from binding to phenylalanine hydroxylase when a competitive inhibitor of phenylalanine hydroxylase enzyme activity, 6,7-dimethyl-7,8-dihydropterin, was bound to phenylalanine hydroxylase. (c) NS7 antibody was shown to bind to a wide range of pterin-requiring enzymes: phenylalanine, tyrosine and tryptophan hydroxylases, dihydropteridine reductase, dihydrofolate reductase, and sepiapterin reductase. Thus the NS7 antibody has successfully mimicked a common portion of the pterin cofactors utilized by these enzymes and demonstrated structure homology in their pterin binding sites despite their diverse function and little amino acid sequence homology except among the three aromatic amino acid hydroxylases.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Anticuerpos Monoclonales , Dihidropteridina Reductasa/metabolismo , Idiotipos de Inmunoglobulinas , NADH NADPH Oxidorreductasas/metabolismo , Fenilalanina Hidroxilasa/metabolismo , Pteridinas/inmunología , Tetrahidrofolato Deshidrogenasa/metabolismo , Triptófano Hidroxilasa/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Glándulas Suprarrenales/enzimología , Oxidorreductasas de Alcohol/inmunología , Animales , Complejo Antígeno-Anticuerpo/análisis , Sitios de Unión , Bovinos , Dihidropteridina Reductasa/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Hibridomas/inmunología , Cinética , Hígado/enzimología , Fenilalanina Hidroxilasa/inmunología , Ratas , Tetrahidrofolato Deshidrogenasa/inmunología , Triptófano Hidroxilasa/inmunología , Tirosina 3-Monooxigenasa/inmunologíaRESUMEN
We have produced and characterized the first monoclonal antibody against neopterin (D-erythro-6-(1,2,3,-trihydroxypropyl)pterin). The antibody specifically recognizes neopterin in a modified RIA. The binding capacity in this assay is 34%, the sensitivity limit of inhibition is 0.9 nmol/l. Cross-reactivity exists with monapterin (L-threo(1,2,3,trihydroxypropyl)pterin) in 30%, with other pteridines cross-reactivity has been found in less than 5%.