Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

It has become clear that calcium is an important mediator in the transduction of signals due to ligand binding to cell surface receptors. Cytosolic calcium is typically maintained at low levels in both muscle and non-muscle cells and intracellular sequestering of calcium appears to be important in this process. The identification of intracellular calcium pools has been the subject of much recent study, and it has been proposed that such pools would contain three components: a calcium-activated pump or Ca(2+)-ATPase, a calcium channel such as the inositol trisphosphate receptor or ryanodine receptor, and a high-capacity calcium-binding protein such as calsequestrin or calreticulin. We report here on the localization of two components, the organellar Ca(2+)-ATPase (SERCA) and calreticulin, in neuronal tissues. Using immunofluorescence and subcellular fractionation, we have found that for the most part, these two proteins do not co-localize in neuron cell bodies, dendrites, or axons; but may co-localize at the axon terminal.
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PMID:Differences in the subcellular localization of calreticulin and organellar Ca(2+)-ATPase in neurons. 838 14

Caldesmon is known to inhibit actomyosin ATPase and filament sliding in vitro, and may play a role in modulating smooth muscle contraction as well as in diverse cellular processes including cytokinesis and exocytosis. However, the structural basis of caldesmon action has not previously been apparent. We have recorded electron microscope images of negatively stained thin filaments containing caldesmon and tropomyosin which were isolated from chicken gizzard smooth muscle in EGTA. Three-dimensional helical reconstructions of these filaments show actin monomers whose bilobed shape and connectivity are very similar to those previously seen in reconstructions of frozen-hydrated skeletal muscle thin filaments. In addition, a continuous thin strand of density follows the long-pitch actin helices, in contact with the inner domain of each actin monomer. Gizzard thin filaments treated with Ca2+/calmodulin, which dissociated caldesmon but not tropomyosin, have also been reconstructed. Under these conditions, reconstructions also reveal a bilobed actin monomer, as well as a continuous surface strand that appears to have moved to a position closer to the outer domain of actin. The strands seen in both EGTA- and Ca2+/calmodulin-treated filaments thus presumably represent tropomyosin. It appears that caldesmon can fix tropomyosin in a particular position on actin in the absence of calcium. An influence of caldesmon on tropomyosin position might, in principle, account for caldesmon's ability to modulate actomyosin interaction in both smooth muscles and non-muscle cells.
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PMID:Three-dimensional reconstruction of caldesmon-containing smooth muscle thin filaments. 840 15

Recent research has led to an understanding of the in vitro properties of caldesmon, including the regulation of actomyosin ATPase activity, cross-linking between actin and myosin, enhancement of microfilament stability and stimulation of polymerization of actin. While it remains to be established whether caldesmon functions similarly in vivo, recent studies have suggested that smooth muscle caldesmon regulates the inhibition of vascular smooth muscle tone, and that non-muscle caldesmon plays roles in the regulation of cell motility and cytoskeletal organization in three biological activities: granule movement, hormone secretion and reorganization of microfilaments during mitosis.
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PMID:Caldesmon. 844 32

The mechanism by which Bcl-2 inhibits apoptosis is unknown. The Bcl-2 protein is localized to intracellular membranes, including the endoplasmic reticulum (ER). The ER is the major intracellular reservoir of Ca2+ in non-muscle cells, sequestering Ca2+ for use in intracellular signaling, and is a prime target of oxidative damage. Because of the recent suggestion that Bcl-2 acts in an antioxidant pathway, we wondered whether Bcl-2 might protect the ER Ca2+ pool in cells exposed to reactive oxygen species. To test this hypothesis, we assessed the effect of hydrogen peroxide (H2O2) treatment on the ER Ca2+ pool in WEH17.2 cells, which do not express Bcl-2, and two stable transfectants, W.Hb13 and W.Hb12. The Bcl-2 level by Western blotting is 4-fold higher in W.Hb12 cells compared to W.Hb13 cells. The ER Ca2+ pool in H2O2-treated and untreated cells was measured according to the amount of Ca2+ mobilized from the ER lumen into the cytoplasm by thapsigargin (TG), a selective inhibitor of the ER (Ca2+)-ATPase. H2O2 treatment produced a significant reduction in the TG-mobilizable Ca2+ pool in WEH17.2 and W.Hb13 cells, but not in W.Hb12 cells, indicating that overexpression of Bcl-2 preserves the integrity of the ER Ca2+ pool in cells exposed to reactive oxygen species.
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PMID:Bcl-2 inhibits hydrogen peroxide-induced ER Ca2+ pool depletion. 866 30

Ca ATPase regulates intracellular Ca levels by pumping Ca into sarcoplasmic and endoplasmic reticulum (SER). Phospholamban was first identified as a phosphoprotein in cardiac myocytes. Functional properties of phospholamban by steady-state and presteady-state kinetic studies of Ca pump ATPase suggest that phospholamban functions as an inhibitory co-factor for cardiac Ca ATPase (SERCA 2). Protein kinase A-catalyzed phosphorylation of phospholamban results in the dissociation of phospholamban from the Ca ATPase, thus augmenting the ATPase activity. Phospholamban is found as a homo-pentamer, formed from subunits of 6080 Da in size. PKA-catalyzed and CAM kinase- catalyzed phosphorylation residues (Ser 16 and Thr 17) are located in the N-terminal cytoplasmic domain, whereas the C-terminal 22 residues are extremely hydrophobic and are considered to be embedded in the SR membrane. At least three kinds of Ca ATPase have been found. SERCA 1 is expressed in fast-twitch skeletal muscle, while the SERCA 2 gene encodes two alternatively spliced products, SERCA 2a and 2b. SERCA 2a is expressed in cardiac and slow-twitch skeletal muscles; SERCA 2b in smooth muscle and non-muscle tissues. SERCA 3 is expressed in a broad variety of muscle and non-muscle tissues. In vitro expression systems revealed that the functional properties of Ca transport of SERCA 2 are identical to SERCA 1, but not SERCA 3. In particular, the Ca affinity for Ca transport of SERCA 1 or 2 is lowered by co-expression with phospholamban, whereas that of SERCA 3 is not. Identification of the interaction sites of phospholamban and SERCA 2 helps defining the molecular mode of interaction between the two proteins. Photoactivated cross-linking studies indicated that potential binding residues are located just downstream of the active ATPase site (Asp 351) of SERCA 2, but SERCA 3 is devoid of this sequence. If a chimeric Ca ATPase (CH2) is made from SERCA 2 and 3, in which the SERCA 3 region corresponding to the phospholamban-binding sequence of SERCA 2 is introduced into the remainder of the SERCA 2 molecule, then the interaction with phospholamban is lost. These results suggest that this region of SERCA 2 contains amino acids which are involved in the interaction with phospholamban. By site-directed mutagenesis of amino acids of this region, we were able to show that 6 residues, Lys-Asp-Asp-Lys-Pro-Val402, of SERCA 2 are functionally important for the interaction. When the chimera CH2 was mutated back to SERCA 2 type, mutated CH2 containing these 6 residues of SERCA 2 restored the interaction with phospholamban. Altogether, these 6 residues of SERCA 2 represent the interaction sites for phospholamban. Mutagenesis studies of phospholamban also demonstrated that the hydrophilic, cytoplasmic region of phospholamban contains a potential binding site for SERCA 2. We therefore conclude that the functional interaction between the two proteins occurs in the cytoplasmic region.
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PMID:SR Ca(2+)-ATPase/phospholamban in cardiomyocyte function. 895 64

Regulation of a variety of cellular contractile events requires that vertebrate smooth and non-muscle myosin II can achieve an "off" state. To examine the role of the myosin rod in this process, we determined the minimal size at which a myosin molecule is capable of regulation via light chain phosphorylation. Expressed smooth muscle myosin subfragments with as many as 100 amino acids of the coiled-coil rod sequence did not dimerize and were active independently of phosphorylation. To test whether dimerization per se restores regulation of ATPase activity, mutants were expressed with varying lengths of rod sequence, followed by C-terminal leucine zippers to stabilize the coiled-coil. Dimerization restored partial regulation, but the presence of a length of rod approximately equal to the myosin head was necessary to achieve a completely off state. Partially regulated short dimers could be converted into fully regulated molecules by addition of native rod sequence after the zipper. These results suggest that the myosin rod mediates specific interactions with the head that are required to obtain the completely inactive state of vertebrate smooth and non-muscle myosins. If these interactions are prohibited under cellular conditions, unphosphorylated crossbridges can slowly cycle.
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PMID:Spare the rod, spoil the regulation: necessity for a myosin rod. 899 Jan 59

We have isolated a novel, high Mr protein from human retinal pigment epithelial cells and endothelial cells by affinity chromatography on Sepharose 4B. Two polypeptides are present on SDS-gels of the 8 M urea eluent with apparent molecular mass of approximately 210 and 47 kDa. In the absence of dithiothreitol, the two polypeptides migrate as one protein band with an apparent molecular mass of approximately 550 kDa. "Piglet," as this molecule is tentatively named, is present in retinal pigment epithelial and endothelial cells of several species, but could not be detected in the nonepithelial cells we examined. Immunofluorescent localization using an antibody to the 210-kDa polypeptide revealed a filamentous network in the cytoplasm of cultured cells. This antibody was used to identify a cDNA for piglet in a bovine aortic endothelial cell expression library. Sequence data indicate a high degree of identity with non-muscle myosin II heavy chain. We subsequently found that piglet had an actin-activated ATPase activity, colocalized with actin in cells, and reacted on Western blots with a pan-non-muscle myosin II heavy chain antiserum. The protein was also recognized by antibodies specific for myosin heavy chain isoform A, but did not react with anti-isoform B antibodies. Although piglet has several features in common with known forms of non-muscle myosin II, the distinctly unconventional features it displays suggest that it is a novel myosin.
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PMID:Novel characteristics of a myosin isolated from mammalian retinal pigment epithelial and endothelial cells. 907 11

Escherichia coli dimethyl sulfoxide reductase is a heterotrimer comprising a catalytic subunit (DmsA), an electron transfer subunit (DmsB) and an integral membrane anchor subunit (DmsC). DmsC is not antigenic and the production of antibodies to this subunit has not been successful. We have tagged DmsC at the C-terminus with a dystrophin-specific amino acid sequence (dysp) to which antibodies are readily available. We were able to use this tagging technique to monitor expression and localization of DmsC in E. coli and non-muscle eukaryotic cells. Growth properties of wild-type E. coli, strain HB101, overexpressing DmsC:dysp suggest that the expression of DmsC is lethal to E. coli. The lethality could be overcome by utilizing an E. coli F0F1 ATPase mutant as the host. Growth conditions of culture density, duration of induction, temperature of incubation after induction and media conditions were investigated to optimize DmsC:dysp accumulation levels. In order to alleviate the problem arising from the toxicity of DmsC, expression in eukaryotic tissue culture was also explored. A plasmid expressing DmsC:dysp was transfected into COS-1 or McA-RH777 cells. The presence of expressed DmsC:dysp was confirmed using specific anti-dysp antibodies and immunofluorescence microscopy analysis revealed that the DmsC:dysp was localized to the endoplasmic reticulum. Expression of DmsC:dysp did not appear to be toxic to the eukaryotic cells. These data suggest methodologies to overcome lethality problems associated with the overexpression of integral membrane proteins like DmsC.
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PMID:Expression and epitope tagging of the membrane anchor subunit (DmsC) of Escherichia coli dimethyl sulfoxide reductase. 915 79

Caldesmon inhibits actomyosin ATPase and filament sliding in vitro, and therefore may play a role in modulating smooth and non-muscle motile activities. A bacterially expressed caldesmon fragment, 606C, which consists of the C-terminal 150 amino acids of the intact molecule, possesses the same inhibitory properties as full-length caldesmon and was used in our structural studies to examine caldesmon function. Three-dimensional image reconstruction was carried out from electron micrographs of negatively stained, reconstituted thin filaments consisting of actin and smooth muscle tropomyosin both with and without added 606C. Helically arranged actin monomers and tropomyosin strands were observed in both cases. In the absence of 606C, tropomyosin adopted a position on the inner edge of the outer domain of actin monomers, with an apparent connection to sub-domain 1 of actin. In 606C-containing filaments that inhibited acto-HMM ATPase activity, tropomyosin was found in a different position, in association with the inner domain of actin, away from the majority of strong myosin binding sites. The effect of caldesmon on tropomyosin position therefore differs from that of troponin on skeletal muscle filaments, implying that caldesmon and troponin act by different structural mechanisms.
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PMID:Three-dimensional image reconstruction of reconstituted smooth muscle thin filaments: effects of caldesmon. 916 17

Sarco(endo)plasmic reticulum Ca2+ ATPase isoform 3 (SERCA3) is one of two Ca2+ pumps serving intracellular Ca2+ signaling pools in non-muscle tissues; however, unlike the ubiquitous SERCA2b, it exhibits a restricted cell-type distribution. Gene targeting was used to generate a mouse with a null mutation in the SERCA3 gene. Homozygous mutant mice were viable, fertile, and did not exhibit an overt disease phenotype. Because SERCA3 is expressed in arterial endothelial cells, aortic ring preparations were analyzed to determine whether it is involved in the regulation of vascular tone. Contraction-isometric force relations in response to phenylephrine or KCl, as well as relaxation produced by exposure to a nitric oxide donor, were similar in wild-type and null mutant aortas. Acetylcholine-induced endothelium-dependent relaxation of aortas after precontraction with phenylephrine was significantly reduced in homozygous mutants (61.3 +/- 5.6% in wild type, 35.4 +/- 7.3% in mutants). Ca2+ imaging of cultured aortic endothelial cells demonstrated that the acetylcholine-induced intracellular Ca2+ signal is sharply diminished in SERCA3-deficient cells and also indicated that replenishment of the acetylcholine-responsive Ca2+ stores is severely impaired. These results indicate that SERCA3 plays a critical role in endothelial cell Ca2+ signaling events involved in nitric oxide-mediated relaxation of vascular smooth muscle.
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PMID:Defective endothelium-dependent relaxation of vascular smooth muscle and endothelial cell Ca2+ signaling in mice lacking sarco(endo)plasmic reticulum Ca2+-ATPase isoform 3. 937 48


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