Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.6.1.3 (ATPase)
 65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We recently identified a multi-SERCA (sarco/endoplasmic reticulum Ca2+ ATPase) system in haemopoietic cells comprising the SERCA 2b, SERCA 3 and a new monoclonal anti-Ca2+ ATPase antibody (PL/IM 430) recognizable SERCA isoforms. We have now investigated the subcellular localization of these enzymes in human platelets by Western blotting of subcellular membrane fractions and by immunoelectron microscopy. We precisely defined the recognition specificity of the polyclonal anti-SERCA 2b, anti-SERCA 3, anti-SERCA 1 antibodies as well as of the monoclonal antibody PL/IM 430 by testing their recognition of the tryptic fragments of the SERCA isoforms. The analysis of fragmented membranes enriched in plasma membrane and intracellular membrane components by Western blotting showed that the SERCA 2b and the SERCA 3 isoforms were found in both the plasma membrane and the intracellular membrane fractions, whereas the PL/IM 430 recognizable SERCA isoform was restricted to membranes associated with the plasma membrane fraction. The immunoelectron microscopical study of the SERCA isoforms in resting platelets showed that: (i) the SERCA 2b isoform was expressed in membranes associated with the plasma membrane and open canalicular system, some alpha-granules and in unidentified membranes; (ii) the SERCA 3 isoform was found associated with plasma and intracellular membranes; and (iii) the PL/IM 430 recognizable SERCA isoform was observed only in structures associated with the cytoplasmic face of the plasma membranes, as confirmed by flow cytometry. Finally, since the PL/IM 430 antibody was raised against intracellular membranes, we looked for a potential membrane redistribution during the isolation procedure used for the preparation of the immunizing membranes. Neuraminidase treatment indeed induced a translocation of the PL/IM 430 recognizable SERCA isoform from plasma to intracellular membranes. Thus, the multi-SERCA system in platelets: (i) is distributed over different platelet membranes, (ii) presents a sub-compartmental organization with some overlapping, and (iii) is partly associated with motile membranes, reflecting an unrecognized level of complexity of Ca2+ stores in these cells.
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PMID:Immunolocalization of the multi-sarco/endoplasmic reticulum Ca2+ ATPase system in human platelets. 913 65

The endoplasmic reticulum (ER) plays a pivotal role in the regulation of cytosolic Ca(2+) concentrations ([Ca(2+)](cyt)) and hence in insulin secretion from pancreatic beta-cells. However, the molecular mechanisms involved in both the uptake and release of Ca(2+) from the ER are only partially defined in these cells, and the presence and regulation of ER ryanodine receptors are a matter of particular controversy. To monitor Ca(2+) fluxes across the ER membrane in single live MIN6 beta-cells, we have imaged changes in the ER intralumenal free Ca(2+) concentration ([Ca(2+)](ER)) using ER-targeted cameleons. Resting [Ca(2+)](ER) (approximately 250 micromol/l) was markedly reduced after suppression (by approximately 40%) of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)-2b but not the SERCA3 isoform by microinjection of antisense oligonucleotides, implicating SERCA2b as the principle ER Ca(2+)-ATPase in this cell type. Nutrient secretagogues that elevated [Ca(2+)](cyt) also increased [Ca(2+)](ER), an effect most marked at the cell periphery, whereas inositol 1,4,5-trisphosphate-generating agents caused a marked and homogenous lowering of [Ca(2+)](ER). Demonstrating the likely presence of ryanodine receptors (RyRs), caffeine and 4-chloro-3-ethylphenol both caused an almost complete emptying of ER Ca(2+) and marked increases in [Ca(2+)](cyt). Furthermore, photolysis of caged cyclic ADP ribose increased [Ca(2+)](cyt), and this effect was largely abolished by emptying ER/Golgi stores with thapsigargin. Expression of RyR protein in living MIN6, INS-1, and primary mouse beta-cells was also confirmed by the specific binding of cell-permeate BODIPY TR-X ryanodine. RyR channels are likely to play an important part in the regulation of intracellular free Ca(2+) changes in the beta-cell and thus in the regulation of insulin secretion.
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PMID:Dynamic imaging of endoplasmic reticulum Ca2+ concentration in insulin-secreting MIN6 Cells using recombinant targeted cameleons: roles of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)-2 and ryanodine receptors. 1181 80

Paxilline, an indole alkaloid mycotoxin from Penicillium paxilli, is an inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA). Paxilline inhibited differing isoforms of SERCA with IC50s between 5 and 50 microM. It inhibited more potently the purified Ca2+ ATPase activity from skeletal muscle with an IC50 of 5 microM. Detailed effects of this inhibitor on the Ca2+ and ATP dependence upon activity indicate that it affects the high-affinity Ca2+-binding (E1) form of the ATPase. In addition, paxilline is a "competitive" inhibitor with respect to high concentrations of ATP, increasing the regulatory binding site K(m), without affecting the catalytic binding site K(m). At higher concentrations, paxilline inhibits phosphoenzyme formation from ATP and inorganic phosphate, without affecting nucleotide binding. We therefore suggest that paxilline has two effects on the Ca2+ ATPase. At lower concentrations (5-10 microM), paxilline inhibits the ATP-dependent acceleration of Ca2+ release from the phosphoenzyme and/or phosphoenzyme decay. At higher concentrations, paxilline inhibits phosphoenzyme formation.
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PMID:The mechanism of inhibition of the sarco/endoplasmic reticulum Ca2+ ATPase by paxilline. 1223 90

Since Ca(2+)-ATPase is a major determinant of calcium homeostasis in the lens, we examined the expression of Ca(2+)-ATPase by calcium. An immortalized human lens epithelial cell line, HLE B-3, was treated with thapsigargin to inhibit sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) releasing calcium from intracellular stores. Isoforms of the plasma membrane Ca(2+)-ATPase (PMCA) and SERCA were quantified by Western blot and quantitative real time reverse transcription polymerase chain reaction. We showed that both PMCA1 and SERCA3 isoform protein and mRNA are upregulated two- to three-fold in thapsigargin-treated HLE B-3 cells in a time and dose-dependent manner. Thapsigargin did not change the protein or mRNA levels of PMCA2, 3, 4 or SERCA2b. Considering the harmful effects of increased intracellular calcium levels, the upregulation of both SERCA and PMCA pumps suggests it is a compensatory mechanism to restore the calcium concentration to the physiological resting level.
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PMID:Regulation of sarco/endoplasmic and plasma membrane calcium ATPase gene expression by calcium in cultured human lens epithelial cells. 1687 31

Agonist-sensitive intracellular Ca2+ stores may be heterogeneous and exhibit distinct functional features. We have studied the properties of intracellular Ca2+ stores using targeted aequorins for selective measurements in different subcellular compartments. Both, HEK-293T [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] and HeLa cells accumulated Ca2+ into the ER (endoplasmic reticulum) to near millimolar concentrations and the IP3-generating agonists, carbachol and ATP, mobilized this Ca2+ pool. We find in HEK-293T, but not in HeLa cells, a distinct agonist-releasable Ca2+ pool insensitive to the SERCA (sarco/endoplasmic reticulum Ca2+ ATPase) inhibitor TBH [2,5-di-(t-butyl)-benzohydroquinone]. TG (thapsigargin) and CPA (cyclopiazonic acid) completely emptied this pool, whereas lysosomal disruption or manoeuvres collapsing endomembrane pH gradients did not. Our results indicate that SERCA3d is important for filling the TBH-resistant store as: (i) SERCA3d is more abundant in HEK-293T than in HeLa cells; (ii) the SERCA 3 ATPase activity of HEK-293T cells is not fully blocked by TBH; and (iii) the expression of SERCA3d in HeLa cells generated a TBH-resistant agonist-mobilizable compartment in the ER. Therefore the distribution of SERCA isoforms may originate the heterogeneity of the ER Ca2+ stores and this may be the basis for store specialization in diverse functions. This adds to recent evidence indicating that SERCA3 isoforms may subserve important physiological and pathophysiological mechanisms.
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PMID:Two distinct calcium pools in the endoplasmic reticulum of HEK-293T cells. 2121 19