RESUMEN
We have previously demonstrated that Na+, K(+)-ATPase activity is present in both differentiated plasma membranes from Electrophorus electricus (L.) electrocyte. Considering that the alpha subunit is responsible for the catalytic properties of the enzyme, the aim of this work was to study the presence and localization of alpha isoforms (alpha1 and alpha2) in the electrocyte. Dose-response curves showed that non-innervated membranes present a Na+, K(+)-ATPase activity 2.6-fold more sensitive to ouabain (I50=1.0+/-0.1 microM) than the activity of innervated membranes (I50=2.6+/-0.2 microM). As depicted in [3H]ouabain binding experiments, when the [3H]ouabain-enzyme complex was incubated in a medium containing unlabeled ouabain, reversal of binding occurred differently: the bound inhibitor dissociated 32% from Na+, K(+)-ATPase in non-innervated membrane fractions within 1 h, while about 50% of the ouabain bound to the enzyme in innervated membrane fractions was released in the same time. These data are consistent with the distribution of alpha1 and alpha2 isoforms, restricted to the innervated and non-innervated membrane faces, respectively, as demonstrated by Western blotting from membrane fractions and immunohistochemical analysis of the main electric organ. The results provide direct evidence for a distinct distribution of Na+, K(+)-ATPase alpha-subunit isoforms in the differentiated membrane faces of the electrocyte, a characteristic not yet described for any polarized cell.
Asunto(s)
Electrophorus/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Western Blotting , Fraccionamiento Celular , Membrana Celular/química , Membrana Celular/enzimología , Polaridad Celular , Órgano Eléctrico/enzimología , Microscopía Confocal , Proteínas Musculares , Ouabaína/metabolismo , Ouabaína/farmacología , Unión Proteica , Isoformas de Proteínas/análisis , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidoresRESUMEN
The effects of tricyclic antidepressants drugs (TCA) amitriptyline, imipramine and nortriptyline, on purified Electrophorus electricus (L.) acetylcholinesterase (AChE; acetylcholine hydrolase, EC 3.1.1.7) were studied using kinetic methods and specific fluorescent probe propidium. The antidepressants inhibited AChE activity by a non-competitive mechanism. Inhibition constants range from 200 to 400 microM. Dimethylated amitriptyline and imipramine were more potent inhibitors than the monomethylated nortriptyline. Fluorescence measurements using bis-quaternary ligand propidium were used to monitor ligand-binding properties of these cationic antidepressants to the AChE peripheral anionic site (PAS). This ligand exhibited an eight-fold fluorescence enhancement upon binding to the peripheral anionic site of AChE from E. electricus (L.) with K(D)=7 x 10(-7)M. It was observed that TCA drugs displaced propidium from the enzyme. On the basis of the displacement experiments antidepressant dissociation constants were determined. Similar values for the inhibition constants suggest that these drugs have similar affinity to the peripheral anionic site. The results also indicate that the catalytic active center of AChE does not participate in the interaction of enzyme with tricyclic antidepressants. These studies suggest that the binding site for tricyclic antidepressants is located at the peripheral anionic site of E. electricus (L.) acetylcholinesterase.
Asunto(s)
Acetilcolinesterasa/metabolismo , Antidepresivos Tricíclicos/farmacología , Inhibidores de la Colinesterasa/metabolismo , Electrophorus/metabolismo , Amitriptilina/farmacología , Animales , Colorantes/metabolismo , Imipramina/farmacología , Estructura Molecular , Nortriptilina/farmacología , Propidio/metabolismo , Unión Proteica , Espectrometría de FluorescenciaRESUMEN
Acetylcholine is the neurotransmitter responsible for the transmission of impulses from cholinergic neurons to cells of innervated tissues. Its biosynthesis is catalyzed by the enzyme Choline acetyltransferase that is considered to be a phenotypically specific marker for cholinergic system. It is well known that the regulation of Choline acetyltransferase activity under physiological and pathological conditions is important for development and neuronal activities of cholinergic functions. We observed the distribution of Choline acetyltransferase in sections from the normal and denervated main electric organ sections of Electrophorus electricus (L.) by immunofluorescence using a anti-Choline acetyltransferase antibody. The animals were submitted to a surgical procedure to remove about 20 nerves and after 30 and 60 days, they were sacrificed. After 30 days, the results from immunohistochemistry demonstrated an increase on the Choline acetyltransferase distribution at denervated tissue sections when compared with the sections from the normal contralateral organ. A very similar labeling was observed between normal and denervated tissue sections of the animals after 60 days. However, Choline acetyltransferase activity (nmolesACh/ min/ mg of protein) in extracts obtained from electrocyte microsomal preparation, estimated by Fonnun's method (Fonnun 1975), was 70% lower in the denervated extracts.
Asunto(s)
Colina O-Acetiltransferasa/análisis , Desnervación , Electrophorus/metabolismo , Animales , Biomarcadores/análisis , Microscopía Confocal/métodosRESUMEN
The present investigation deals with the purification and the partial characterization of the soluble creatine kinase (CK) isoenzyme, isolated from the electric organ electrocyte of Electrophorus electricus (L.). Purification was performed by precipitation of the enzyme in the crude extract with ammonium sulfate (80%). The precipitate obtained was analyzed on an ion exchange column of diethylaminoethyl cellulose-52 (DEAE) followed by gel filtration on Superose 12 in a Fast Protein Liquid Chromatography (FPLC) system. Electrophoretic mobility of the active peak confirmed previous results identifying the hybrid isoenzyme MB in the electrocyte cytoplasm. Electrocyte CK is a dimeric enzyme with two identical subunits of approximately 40 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The sequence analysis of the N-terminal peptide (14 amino acids) of the 40 kDa subunit showed homology with other CK enzymes from electric fish (Torpedo) and human muscle type CK.