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
Plasma membrane Ca2+-ATPase activity diminishes by about 50% in red blood cells during preeclampsia. We investigated whether the number of Ca2+-ATPase molecules is modified in red cell membranes from preeclamptic pregnant women by measuring the specific phosphorylated intermediate of this enzyme. Also, we isolated the Ca2+-ATPase protein from both normotensive and preeclamptic pregnant women and estimated its molecular weight, and its cross-reactions with specific polyclonal and monoclonal (5F10) antibodies against it. We measured the Ca2+-ATPase activity in a purified state and the effect of known modulators of this ATPase. It was found that the phosphorylated intermediate associated with PMCA is similar for red cell ghosts from normotensive and preeclamptic women, suggesting a similar number of ATPase molecules in these membranes. The molecular weight of the Ca2+-ATPase is around 140 kDa for both normotensive and preeclamptic membranes, and its cross-reactions with specific antibodies is similar, suggesting that the protein structure remains intact in preeclampsia. Calmodulin, ethanol, or both calmodulin plus ethanol, stimulated the Ca2+-ATPase activity to the same extent for both normotensive and preeclamptic preparations. Our results showed that the reduced Ca2+-ATPase activity of the red cell membranes from preeclamptic women is not associated with a defective enzyme, but rather with a high level of lipid peroxidation.
Asunto(s)
ATPasas Transportadoras de Calcio/metabolismo , Proteínas de Transporte de Catión/metabolismo , Membrana Eritrocítica/enzimología , Preeclampsia/enzimología , Adolescente , Adulto , Anticuerpos/inmunología , Presión Sanguínea/fisiología , ATPasas Transportadoras de Calcio/química , ATPasas Transportadoras de Calcio/inmunología , Calmodulina/metabolismo , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/inmunología , Etanol/metabolismo , Femenino , Humanos , Peroxidación de Lípido , Peso Molecular , Placenta/metabolismo , Placenta/patología , ATPasas Transportadoras de Calcio de la Membrana Plasmática , Preeclampsia/sangre , Embarazo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
The purified plasma membrane Ca(2+) pump (PMCA) was digested with trypsin, and the proteolytic products were identified by immunoblotting with monoclonal antibodies JA9 or 5F10 directed against the extreme N-terminal segment and the central portion of the molecule, respectively. After a short treatment with low concentrations of the protease, JA9 reacted predominantly with a peptide of 35 kDa whereas 5F10 detected a peptide of 90 kDa. The trypsin cut leading to the production of these fragments had no effect on the maximal activity of the enzyme. At higher concentrations of trypsin, JA9 detected a main fragment of 33 kDa and smaller fragments of 19 and 15 kDa. The persistence of fragments reacting with JA9 indicates that the N-terminal region containing its epitope (residues 51-75) was not easily accessible to the protease in the native PMCA. However, the reactivity with JA9 was rapidly lost during proteolysis of the denatured protein. The passage of the mixture of PMCA fragments through a calmodulin-Sepharose column resulted in the retention of the N-terminal 35 kDa fragment together with that of 90 kDa, despite the fact that only the latter binds calmodulin. The ethylenediaminetetraacetic acid (EDTA) eluate, which contained about equal amounts of both fragments, had a Ca(2+) ATPase activity similar to that of the intact enzyme. The tight association between the two peptides was evidenced by the fact that concentrations of polyoxyethylene 10 lauryl ether (C(12)E(10)), sodium dodecyl sulfate (SDS) high enough for inactivating the enzyme and dissociate the pump from calmodulin were unable of breaking the interaction between the 35 and 90 kDa fragments. Altogether, these results show that after digestion with trypsin, the N-terminal portion of the PMCA, including the extreme N-terminal segment, remains part of a fully functional catalytic complex.