RESUMEN
In higher eukaryotes, the sarco-endoplasmic reticulum (ER) Ca(2+)-ATPase (SERCA) is characterized for its high sensitivity to low concentrations of thapsigargin (TG), a very specific inhibitor. In contrast, SERCA-like enzymes with different sensitivities to TG have been reported in trypanosomatids. Here, we characterized a SERCA-like enzyme from Trypanosoma evansi and evaluated its interaction with TG. Confocal fluorescence microscopy using BODIPY FL TG and specific anti-SERCA antibodies localized the T. evansi SERCA-like enzyme in the ER and confirmed its direct interaction with TG. Moreover, the use of either 1 µM TG or 25 µM 2',5'-di (tert-butyl)-1,4-benzohydroquinone prevented the reuptake of Ca(2+) and consequently produced a small increase in the parasite cytosolic calcium concentration in a calcium-free medium, which was released from the ER pool. A 3035 bp-sequence coding for a protein with an estimated molecular mass of 110.2 kDa was cloned from T. evansi. The corresponding gene product contained all the invariant residues and conserved motifs found in other P-type ATPases but lacked the calmodulin binding site. Modeling of the three-dimensional structure of the parasite enzyme revealed that the amino acid changes found in the TG-SERCA binding pocket do not compromise the interaction between the enzyme and the inhibitor. Therefore, we concluded that T. evansi possesses a SERCA-like protein that is inhibited by TG.
Asunto(s)
ATPasas Transportadoras de Calcio/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Bombas Iónicas/efectos de los fármacos , Tapsigargina/farmacología , Trypanosoma/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , ATPasas Transportadoras de Calcio/genética , ATPasas Transportadoras de Calcio/inmunología , Retículo Endoplásmico/enzimología , Enfermedades de los Caballos/parasitología , Caballos , Bombas Iónicas/metabolismo , Masculino , Microscopía Confocal , Modelos Moleculares , Datos de Secuencia Molecular , Ratas , Ratas Sprague-Dawley , Alineación de Secuencia , Trypanosoma/efectos de los fármacos , Trypanosoma/fisiología , Tripanosomiasis/parasitología , Tripanosomiasis/veterinariaRESUMEN
A microsomal fraction enriched in ion pump enzymes was isolated from the gill of the carp (Cyprinus carpio Linneo). Mg(2+)-dependent (Na+ + K+), Na+, HCO3- and Ca(2+)-stimulated ATPase activities were studied following treatment with microcystin-LR-like toxin, the major toxic component isolated from Microcystis aeruginosa culture. These enzyme activities were inhibited in a dose-dependent manner. The maximum inhibition of each enzyme, induced with nM concentration of the toxin, was similar to that produced by inhibitors specific for each ATPase activity. The Mg(2+)-ATPase activity and non-specific hydrolysis of ATP were unaffected. These results strongly suggest that the massive fish death during M. aeruginosa blooms may result from the loss of ion homeostatic processes produced by the inhibitory action of microcystin on the ion pumps of gill chloride cells.
Asunto(s)
Toxinas Bacterianas/farmacología , Branquias/enzimología , Bombas Iónicas/efectos de los fármacos , Microcystis/química , Animales , ATPasas Transportadoras de Calcio/metabolismo , Carpas , Branquias/microbiología , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
1. Na(+)o-dependent Ca2+ efflux (forward Na(+)-Ca2+ exchange), and in some cases the Na(+)i-dependent Ca2+ influx (reverse Na(+)-Ca2+ exchange) were measured in internally dialysed squid axons under membrane potential control. 2. We tested the effect on the Na(+)-Ca2+ exchange of the MgATP analogue bidentate chromium adenosine-5'-triphosphate (CrATP), substrate of several kinases, and cobalt tetrammine ATP (Co(NH3)4ATP), a poor substrate of most kinases. 3. CrATP completely blocked the MgATP and MgATP-gamma-S (ATP-gamma-S) stimulation of the Na(+)o-dependent Ca2+ efflux (forward exchange) and the Na+i-dependent Ca2+ influx (reverse exchange). The analogue only blocked the nucleotide-dependent fraction of the Na(+)-Ca2+ exchange without modifying any kinetic parameters of the exchange reactions. 4. The effects of CrATP were fully reversible with a very slow time constant (t 1/2 about 30 min). 5. The MgATP stimulation of the Na(+)-Ca2+ exchange was completely saturated at 1 mM. Higher MgATP concentrations (up to 15 mM) had no additional effects. Pentalysine (internal or external), the protein kinase C inhibitor H-7 (1-(5-isoquinolinylsulphonyl)-2-methylpiperazine) and several calmodulin inhibitors did not inhibit Na(+)-Ca2+ exchange either in the absence or presence of MgATP. 6. Our results do not agree with the idea of an aminophospholipid translocase being responsible for the ATP stimulation of the Na(+)-Ca2+ exchange in squid axons; they suggest that this is due to the action of a kinase system.