RESUMEN
Early physiological and pharmacological studies of crayfish and squid giant nerve fibers suggested that glutamate released from the axon during action potential generation initiates metabolic and electrical responses of periaxonal glia. However, more recent investigations in our laboratories suggest that N-acetylaspartylglutamate (NAAG) may be the released agent active at the glial cell membrane. The investigation described in this paper focused on NAAG metabolism and release, and its contribution to the appearance of glutamate extracellularly. Axoplasm and periaxonal glial cell cytoplasm collected from medial giant nerve fibers (MGNFs) incubated with radiolabeled L-glutamate contained radiolabeled glutamate, glutamine, NAAG, aspartate, and GABA. Total radiolabel release was not altered by electrical stimulation of nerve cord loaded with [(14)C]glutamate by bath application or loaded with [(14)C]glutamate, [(3)H]-D-aspartate or [(3)H]NAAG by axonal injection. However, when radiolabeled glutamate was used for bath loading, radiolabel distribution among glutamate and its metabolic products in the superfusate was changed by stimulation. NAAG was the largest fraction, accounting for approximately 50% of the total recovered radiolabel in control conditions. The stimulated increase in radioactive NAAG in the superfusate coincided with its virtual clearance from the medial giant axon (MGA). A small, stimulation-induced increase in radiolabeled glutamate in the superfusate was detected only when a glutamate uptake inhibitor was present. The increase in [(3)H]glutamate in the superfusion solution of nerve incubated with [(3)H]NAAG was reduced when beta-NAAG, a competitive glutamate carboxypeptidase II (GCP II) inhibitor, was present.Overall, these results suggest that glutamate is metabolized to NAAG in the giant axon and its periaxonal glia and that, upon stimulation, NAAG is released from the axon and converted in part to glutamate by GCP II. A quisqualate- and beta-NAAG-sensitive GCP II activity was detected in nerve cord homogenates. These results, together with those in the accompanying paper demonstrating that NAAG can activate a glial electrophysiological response comparable to that initiated by glutamate, implicate NAAG as a probable mediator of interactions between the MGA and its periaxonal glia.
Asunto(s)
Astacoidea/metabolismo , Axones/metabolismo , Comunicación Celular/fisiología , Dipéptidos/biosíntesis , Sistema Nervioso/metabolismo , Neuroglía/metabolismo , Transducción de Señal/fisiología , Animales , Ácido Aspártico/metabolismo , Astacoidea/citología , Astacoidea/efectos de los fármacos , Axones/efectos de los fármacos , Radioisótopos de Carbono/metabolismo , Carboxipeptidasas/efectos de los fármacos , Carboxipeptidasas/metabolismo , Comunicación Celular/efectos de los fármacos , Medios de Cultivo/química , Medios de Cultivo/farmacología , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Dipéptidos/metabolismo , Dipéptidos/farmacocinética , Estimulación Eléctrica , Glutamato Carboxipeptidasa II , Ácido Glutámico/metabolismo , Ácido Glutámico/farmacocinética , Glutamina/metabolismo , Sistema Nervioso/citología , Sistema Nervioso/efectos de los fármacos , Neuroglía/efectos de los fármacos , Técnicas de Cultivo de Órganos , Ácido Quiscuálico/farmacología , Transducción de Señal/efectos de los fármacos , Tritio/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Glutamate carboxypeptidase II (GCP II) catalyzes the extracellular hydrolysis of the neuromodulator N-acetyl-aspartylglutamate to N-acetyl-aspartate and glutamate. GCP II also hydrolyzes gamma-glutamyl bonds in folylpolyglutamate. The predicted amino acid sequence of GCP II displays similarities to aminopeptidases from Streptomyces griseus and Vibrio proteolyticus, whose crystal structures have been determined. These aminopeptidases are cocatalytic zinc metallopeptidases belonging to the peptidase family M28. Specific zinc and substrate ligands have been proposed in GCP II based on the amino acid sequence alignment to these M28 family members. In the present study, site-directed mutagenesis has been used to test the assignment of these putative ligands in human GCP II. Substitutions to the five putative zinc ligands resulted in severely reduced enzyme activity, although mutant protein was expressed as demonstrated by immunoblot analysis. In addition, substitutions of amino acids near the putative zinc ligands have identified other specific residues important for enzyme structure and/or function. Substitutions to putative substrate ligands were less perturbing, and increases in Km were observed for substitutions that introduced a large charge perturbation (e.g., Lys to Glu). The results from substitutions at the proposed zinc and substrate ligands are consistent with the assignment of these residues and suggest that GCP II has a three-dimensional structure similar to other members of the peptidase family M28.
Asunto(s)
Antígenos de Superficie , Carboxipeptidasas/química , Sitios de Unión , Carboxipeptidasas/fisiología , Glutamato Carboxipeptidasa II , Humanos , Immunoblotting , Mutagénesis Sitio-Dirigida , Relación Estructura-Actividad , Zinc/metabolismoRESUMEN
N-Acetylated alpha-linked acidic dipeptidase (NAALADase) is a neuropeptidase that may modulate glutamatergic neurotransmission. Independent of its characterization in the nervous system, one form of NAALADase was shown to be expressed at high levels in human prostatic adenocarcinomas, and it was designated the prostate-specific membrane antigen (PSMA). The NAALADase/PSMA gene is known to produce multiple mRNA splice forms, and based on previous immunohistochemical evidence, it had been assumed that the human brain and prostate expressed different isoforms of the enzyme. Because PSMA is being actively pursued as a target for autoimmune and cytotoxic targeting strategies to treat prostate cancer, the rigorous comparison of the two forms of the enzyme remained an important but untested question. To assess similarities and/or differences between human brain NAALADase and PSMA, we compared the two molecules using criteria of activity, immunoreactivity and sequences of the corresponding mRNAs. NAALADase from human cerebellar isolates displayed a kinetic profile and pharmacological sensitivities similar to PSMA. Also, Northern hybridization to PSMA cDNA detected indistinguishable sets of 2.8-, 4.0- and 6.0-kb RNA species in human brain and the LNCaP prostatic tumor cell line. In addition, the monoclonal antibody 7E11-C5 directed against the prostatic form of the enzyme immunoprecipitated 82% of human cerebellar NAALADase activity. Moreover, reverse transcription-polymerase chain reaction cloning of cerebellar cDNAs indicated that the human brain and prostate express a common mRNA splice form. Therefore, we conclude that the form of NAALADase also known as PSMA is expressed in brain and comprises a significant fraction of brain NAALADase activity.
Asunto(s)
Antígenos de Superficie/metabolismo , Encéfalo/enzimología , Carboxipeptidasas/metabolismo , Northern Blotting , Línea Celular , Clonación Molecular , Glutamato Carboxipeptidasa II , Humanos , Cinética , Reacción en Cadena de la Polimerasa , Pruebas de Precipitina , ARN Mensajero/biosíntesis , ARN Mensajero/químicaRESUMEN
Glutamate carboxypeptidase II may modulate excitatory neurotransmission through the catabolism of the neuropeptide N-acetylaspartylglutamate (NAAG) and possibly other endogenous peptide substrates. To investigate the molecular properties of cloned human GCP II (hGCP II), we analyzed the NAAG-hydrolytic activity conveyed by transfection of a full-length hGCP II cDNA into PC3 cells, which do not express GCP II endogenously. Membrane fractions from these cells demonstrated activity with an apparent Km of 73 nM and Vmax of 35 pmol/(mg protein*min). Activity was inhibited by EDTA and stimulated by the addition of CoCl2. Addition of GCP II inhibitors beta-NAAG, quisqualic acid and 2-(phosphonomethyl)pentanedioic acid (PMPA) inhibited hydrolysis of 2.5 nM NAAG with IC50s of 201 nM, 155 nM and 98 pM, respectively. In competition experiments designed to infer aspects of hGCP II substrate selectivity, NAAG was the most potent alpha peptide tested, with an IC50 of 26 nM. Folate derivatives and some other gamma-glutamyl peptides showed comparable affinity to that of NAAG, also displaying IC50s in the low nM range. Taken together with previous evidence demonstrating their presence in GCP II-expressing tissues, these data suggest that both NAAG and folates are good candidate substrates for GCP II in vivo.
Asunto(s)
Antígenos de Superficie , Carboxipeptidasas/metabolismo , Dipéptidos/metabolismo , Antagonistas de los Receptores Histamínicos H1/metabolismo , Neuropéptidos/metabolismo , Animales , Unión Competitiva/fisiología , Carboxipeptidasas/antagonistas & inhibidores , Carboxipeptidasas/genética , Quelantes/farmacología , Clonación Molecular , Cobalto/farmacología , Cumarinas/farmacología , Ácido Edético/farmacología , Ácido Fólico/análogos & derivados , Ácido Fólico/química , Glutamato Carboxipeptidasa II , Humanos , Hidrólisis , Isocumarinas , Cinética , Leucina/análogos & derivados , Leucina/farmacología , Masculino , Pepstatinas/farmacología , Fenantrolinas/farmacología , Neoplasias de la Próstata , Inhibidores de Proteasas/farmacología , Ratas , Inhibidores de Serina Proteinasa/farmacología , Especificidad por Sustrato , Transmisión Sináptica/fisiología , Células Tumorales Cultivadas/enzimologíaRESUMEN
To explore the nature of proposed ligands to the CuA center in cytochrome c oxidase, site-directed mutagenesis has been initiated in subunit II of the enzyme. Mutations were introduced into the mitochondrial gene from the yeast Saccharomyces cerevisiae by high velocity microprojectile bombardment. A variety of single amino acid substitutions at each of the proposed cysteine and histidine ligands (His-161, Cys-196, Cys-200, and His-204 in the bovine numbering scheme), as well as at the conserved Met-207, all result in yeast which fails to grow on ethanol/glycerol medium. Similarly, all possible paired exchange Cys,His and Cys,Met mutants show the same phenotype. Furthermore, protein stability is severely reduced as evidenced by both the absence of an absorbance maximum at 600 nm in the spectra of mutant cells and the underaccumulation of subunit II, as observed by immunolabeling of mitochondrial extracts. In the same area of the protein, a variety of amino acid substitutions at one of the carboxylates previously implicated in binding cytochrome c, Glu-198, allow (reduced) growth on ethanol/glycerol medium, with normal intracellular levels of protein. These results suggest that a precise folding environment of the CuA site within subunit II is essential for assembly or stable accumulation of cytochrome c oxidase in yeast.