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)

Mutants with a cysteine residue in the gamma subunit at position 207 and the epsilon subunit at position 31 were expressed in combination with a c-dimer construct, which contains a single cysteine at position 42 of the second c subunit. These mutants are called gammaY207C/cc'Q42C and epsilonE31C/cc'Q42C, respectively. Cross-linking of epsilon to the c subunit ring was obtained almost to completion without significant effect on any enzyme function, i.e. ATP hydrolysis, ATP synthesis, and ATP hydrolysis-driven proton translocation were all close to that of wild type. The gamma subunit could also be linked to the c subunit ring in more than 90% yield, but this affected coupling. Thus, ATP hydrolysis was increased 2. 5-fold, ATP synthesis was dramatically decreased, and ATP hydrolysis-driven proton translocation was abolished, as measured by the 9-amino-6-chloro-2-methoxyacridinequenching method. These results for epsilonE31C/cc'Q42C indicate that the c subunit ring rotates with the central stalk element. That the gamma-epsilon cross-linked enzyme retains ATPase activity also argues for a gammaepsilon-c subunit rotor. However, the uncoupling induced by cross-linking of gamma to the c subunit ring points to important conformational changes taking place in the gammaepsilon-c subunit interface during this. Blocking these structural changes by cross-linking leads to a proton leak within the F(0).
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PMID:The gammaepsilon-c subunit interface in the ATP synthase of Escherichia coli. cross-linking of the epsilon subunit to the c subunit ring does not impair enzyme function, that of gamma to c subunits leads to uncoupling. 1056 96

The rise in cytosolic Ca(2+) concentration (Ca(2+)(i)) in vascular endothelial cells (ECs) activates the production and release of nitric oxide (NO). NO modifies Ca(2+)(i) homeostasis in many types of nonendothelial cells. However, its effect on endothelial Ca(2+)(i) homeostasis at basal and excited states remains unclear. In the present study, to elucidate the effect of NO on basal Ca(2+)(i), inositol 1,4,5-trisphosphate-induced Ca(2+)(i) release (IICR) was blocked by expressing an antisense against type-1 inositol 1,4,5-trisphosphate receptors or by microinjecting heparin to individual ECs, and the effects of NO that was released by and diffused from adjacent IICR-intact ECs were recorded. After ATP or bradykinin stimulation, IICR-inhibited ECs showed a marked reduction of basal Ca(2+)(i), which was abolished by N(G)-monomethyl-l-arginine monoacetate pretreatment. The reduction disappeared in sparsely seeded ECs. Exogenous NO gas mimicked the effect of ATP or bradykinin to reduce basal Ca(2+)(i). Blocking plasma membrane Ca(2+)-ATPase (PMCA), but not Na(+)-Ca(2+) exchange or sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase, suppressed the reduction, indicating that the reduction resulted from a NO-dependent potentiation of PMCA. To elucidate the effect of NO on elevated Ca(2+)(i), ATP-, bradykinin-, or thapsigargin-evoked Ca(2+)(i) response in the presence and absence of NO production was compared in adjacent IICR-intact ECs. NO was found to potentiate PMCA, which, in turn, greatly attenuated agonist-evoked Ca(2+)(i) elevation. NO also potentiated Ca(2+) influx, which markedly increased the sustained phase of Ca(2+)(i) elevation and possibly NO production. NO did not affect other Ca(2+)(i)-elevating and Ca(2+)(i)-sequestrating components. Thus, NO-dependent potentiation of PMCA is crucial for Ca(2+)(i) homeostasis over a wide Ca(2+)(i) range.
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PMID:Autocrine action and its underlying mechanism of nitric oxide on intracellular Ca2+ homeostasis in vascular endothelial cells. 1085 3

The 100 kDa a-subunit of the yeast vacuolar (H(+))-ATPase (V-ATPase) is encoded by two genes, VPH1 and STV1. These genes encode unique isoforms of the a-subunit that have previously been shown to reside in different intracellular compartments in yeast. Vph1p localizes to the central vacuole, whereas Stv1p is present in some other compartment, possibly the Golgi or endosomes. To compare the properties of V-ATPases containing Vph1p or Stv1p, Stv1p was expressed at higher than normal levels in a strain disrupted in both genes, under which conditions V-ATPase complexes containing Stv1p appear in the vacuole. Complexes containing Stv1p showed lower assembly with the peripheral V(1) domain than did complexes containing Vph1p. When corrected for this lower degree of assembly, however, V-ATPase complexes containing Vph1p and Stv1p had similar kinetic properties. Both exhibited a K(m) for ATP of about 250 microm, and both showed resistance to sodium azide and vanadate and sensitivity to nanomolar concentrations of concanamycin A. Stv1p-containing complexes, however, showed a 4-5-fold lower ratio of proton transport to ATP hydrolysis than Vph1p-containing complexes. We also compared the ability of V-ATPase complexes containing Vph1p or Stv1p to undergo in vivo dissociation in response to glucose depletion. Vph1p-containing complexes present in the vacuole showed dissociation in response to glucose depletion, whereas Stv1p-containing complexes present in their normal intracellular location (Golgi/endosomes) did not. Upon overexpression of Stv1p, Stv1p-containing complexes present in the vacuole showed glucose-dependent dissociation. Blocking delivery of Vph1p-containing complexes to the vacuole in vps21Delta and vps27Delta strains caused partial inhibition of glucose-dependent dissociation. These results suggest that dissociation of the V-ATPase complex in vivo is controlled both by the cellular environment and by the 100-kDa a-subunit isoform present in the complex.
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PMID:Yeast V-ATPase complexes containing different isoforms of the 100-kDa a-subunit differ in coupling efficiency and in vivo dissociation. 1127 48

The rectal gland of the dogfish shark is a model system for active transepithelial transport of chloride. It has been shown previously that mercuric chloride, one of the toxic environmental pollutants, inhibits chloride secretion in this organ. In order to investigate the mechanism of action of HgCl(2) at a membrane-molecular level, plasma membrane vesicles were isolated from the rectal gland and the effect of mercury on the activity of the Na-K-2Cl cotransporter was investigated in isotope flux studies. During a 30 s exposure HgCl(2) inhibited cotransport activity in a dose-dependent manner with an apparent K(i) of approx. 50 microM. The inhibition was complete after 15 s, partly reversible by dilution of the incubation medium and completely attenuated upon addition of reduced glutathione. The extent of inhibition by mercury depended on the ionic composition of the medium. The sensitivity of the cotransporter was highest when only the high affinity binding sites for sodium and chloride were saturated. Organic mercurials such as p-chloromercuribenzoic acid and p-chloromercuriphenylsulfonic acid at 100 microM did not inhibit the cotransporter, similarly exposure of the vesicles to 10 mM H(2)O(2) or 1 mM dithiothreitol for 30 min at 15 degrees C did not change cotransport activity. Transport activity was, however, reduced by 45.9+/-2.5% after an incubation with 3 mM N-ethylmaleimide for 20 min. Blocking free amino groups by N-hydroxysuccinimide or biotinamidocapronate-N-hydroxysulfosuccinimide had no effect. Investigations on the sidedness of the plasma membrane vesicles, employing the asymmetry of the (Na+K)-ATPase, demonstrated a right-side-out orientation in which the former extracellular face of the membrane is exposed to the incubation medium. In addition, extracellular mercury (5x10(-5) M) inhibited bumetanide-sensitive rubidium uptake into T84 cells by 48.5+/-7.1% after a 2 min incubation period. This inhibition was reversible in a manner similar to that observed in the plasma membrane vesicles. These studies suggest that in isolated rectal gland plasma membrane vesicles the Na-K-2Cl cotransporter (sNKCC1) exposes functionally relevant mercury binding sites at its external surface. These sites represent probably cysteines, the accessibility and/or sensitivity of which depends on the functional state of the transporter.
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PMID:Inhibition by mercuric chloride of Na-K-2Cl cotransport activity in rectal gland plasma membrane vesicles isolated from Squalus acanthias. 1134 78

We previously demonstrated that expression of both the alpha1- and alpha2-subunits of Na+-K+-ATPase is elevated after a 2- to 4-day cyclic stretch in aortic smooth muscle cells. In this study, we determined the effect of short-term (2-30 min) cyclic stretch on the activity of the Na pump and investigated possible mechanisms that may be involved in the action of stretch. Na pump activity was significantly increased above the baseline activity between 2 and 30 min of stretch. This effect of stretch was reversible within 1 h. Intracellular Na was also elevated at corresponding time points. Blocking the entry of Na with Gd and amiloride did not affect the stretch-induced increase in Na pump activity. Inhibition of protein kinase A (PKA) activity attenuated the effect of stretch on the Na pump. Furthermore, inhibition of polymerization of actin and phosphatidylinositol 3-kinase (PI3K) activity prevented the action of stretch on Na pump activity. We conclude that the stimulation of the Na pump in response to cyclic stretch requires the integrity of the actin cytoskeleton as well as the activity of PI3K, which has a role in intracellular vesicular trafficking. PKA may also be involved in this effect of stretch on Na pump.
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PMID:Effect of short-term cyclic stretch on sodium pump activity in aortic smooth muscle cells. 1166 68

Energy deficiency and dysfunction of the Na+, K+-ATPase are common consequences of many pathological insults. The nature and mechanism of cell injury induced by impaired Na+, K+-ATPase, however, are not well defined. We used cultured cortical neurons to examine the hypothesis that blocking the Na+, K+-ATPase induces apoptosis by depleting cellular K+ and, concurrently, induces necrotic injury in the same cells by increasing intracellular Ca2+ and Na+. The Na+, K+-ATPase inhibitor ouabain induced concentration-dependent neuronal death. Ouabain triggered transient neuronal cell swelling followed by cell shrinkage, accompanied by intracellular Ca2+ and Na+ increase, K+ decrease, cytochrome c release, caspase-3 activation, and DNA laddering. Electron microscopy revealed the coexistence of ultrastructural features of both apoptosis and necrosis in individual cells. The caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-FMK) blocked >50% of ouabain-induced neuronal death. Potassium channel blockers or high K+ medium, but not Ca2+ channel blockade, prevented cytochrome c release, caspase activation, and DNA damage. Blocking of K+, Ca2+, or Na+ channels or high K+ medium each attenuated the ouabain-induced cell death; combined inhibition of K+ channels and Ca2+ or Na+ channels resulted in additional protection. Moreover, coapplication of Z-VAD-FMK and nifedipine produced virtually complete neuroprotection. These results suggest that the neuronal death associated with Na+, K+-pump failure consists of concurrent apoptotic and necrotic components, mediated by intracellular depletion of K+ and accumulation of Ca2+ and Na+, respectively. The ouabain-induced hybrid death may represent a distinct form of cell death related to the brain injury of inadequate energy supply and disrupted ion homeostasis.
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PMID:Ionic mechanism of ouabain-induced concurrent apoptosis and necrosis in individual cultured cortical neurons. 1185 Apr 62

The mechanism of assisted protein folding by the chaperonin GroEL alone or in complex with the co-chaperonin GroES and in the presence or absence of nucleotides has been subject to extensive investigations during the last years. In this paper we present data where we have inactivated GroEL by stepwise blocking the nucleotide binding sites using the non-hydrolyzable ATP analogue, (Cr(H2O)4)3+ATP. We correlated the amount of accessible nucleotide binding sites with the residual ATP hydrolysis activity of GroEL as well as the residual refolding activity for two different model substrates. Under the conditions used, folding of the substrate proteins and ATP hydrolysis were directly proportional to the residual, accessible nucleotide binding sites. In the presence of GroES, 50% of the nucleotide binding sites were protected from inactivation by CrATP and the resulting protein retains 50% of both ATPase and refolding activity. The results strongly suggest that under the conditions used in our experiments, the nucleotide binding sites are additive in character and that by blocking of a certain number of binding sites a proportional amount of ATP hydrolysis and refolding activities are inactivated. The experiments including GroES suggest that full catalytic activity of GroEL requires both rings of the chaperonin. Blocking of the nucleotide binding sites of one ring still allows function of the second ring.
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PMID:New aspects on the mechanism of GroEL-assisted protein folding. 1200 12

We studied the effects of the neuropeptide oxytocin (OT) on the long-term potentiation (LTP) paradigm in the dentate gyrus (DG) of urethane anesthetized rats. Intracerebroventricular injection of 1 microg of the hormone in 1 microl of physiological solution 2min before tetanization produced a significant decrease in both components of the perforant path evoked potentials (EP) in the DG. The effects appeared right after the tetanization stimuli and were more pronounced in the excitatory postsynaptic components of the EPs. The decrements lasted for the 2h of recording time. We concluded that OT induced and maintained long-term depression on the DG. In contrast, injection of OT in the absence of tetanic stimulation did not significantly affect perforant path EP in the DG. The results are discussed taking particular consideration of the inhibitory effects the OT has on (Ca(2+)+Mg(2+)) ATPase at membrane levels and the potential interference that this action may have with phosphorylation processes via an ectoprotein kinase isolated from membranes of hippocampal pyramidal neurons. Blocking of this ectoprotein kinase in vitro significantly impairs establishment and maintenance of LTP.
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PMID:Oxytocin induces long-term depression on the rat dentate gyrus: possible ATPase and ectoprotein kinase mediation. 1212 11

The engagement of the activating isoforms of C-type lectin inhibitory receptor (CLIR) or killer Ig-like receptor (KIR) by their natural ligands, represented by soluble HLA-I (sHLA-I) molecules, induced programmed cell death of natural killer (NK) cells. Indeed, NK cell apoptosis elicited by either putative HLA-E and HLA-F (sHLA-I non-A, -B, -C, and -G) or sHLA-I-Cw4 or -Cw3 from untransfected or -Cw4 or -Cw3 alleles transfected HLA-A(-), B(-), C(-), G(-), E(+), F(+) 721.221 lymphoblastoid cell line, respectively, was blocked by covering the corresponding activating receptor with either anti-CLIR- or anti-KIR-specific monoclonal antibodies (mAbs). After sHLA-I-activating receptor interaction, NK cells produced and released Fas ligand (FasL), which in turn led to NK cell apoptosis by interacting with Fas at the NK cell surface. Blocking anti-Fas mAb, or anti-FasL mAb, inhibited sHLA-I-mediated apoptosis via activating receptor in NK cell clones. This apoptosis was inhibited by NK cell treatment with cyclosporin A, whereas this drug had no effect on activating receptor-mediated activation of cytolysis. Conversely, concanamycin A, an inhibitor of vacuolar type H(+)-adenosine triphosphatase (H(+)-ATPase) of granules, inhibited activating receptor-induced NK cell cytolysis, suggesting that activating receptor-mediated apoptosis and cytolysis can use different intracellular pathways. Furthermore, a large amount of interferon-gamma (IFN-gamma) was detectable in culture supernatant of activating receptor(+) NK cells incubated with the appropriate sHLA-I ligand. Again, cyclosporin A, but not concanamycin A, strongly reduced activating receptor-mediated IFN-gamma production. This suggests that activating receptor-induced apoptosis of NK cells could play a role in eliminating potentially harmful NK cell clones and, at the same time, it leads to production of IFN-gamma, an antiviral cytokine able to amplify immune responses.
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PMID:Soluble HLA class I induces NK cell apoptosis upon the engagement of killer-activating HLA class I receptors through FasL-Fas interaction. 1239 68

The Na+, K+-ATPase (Na+, K+-pump) plays critical roles in maintaining ion homeostasis. Blocking the Na+, K+-pump may lead to apoptosis. By contrast, whether an apoptotic insult may affect the Na+, K+-pump activity is largely undefined. In cultured cortical neurons, the Na+, K+-pump activity measured as a membrane current Ipump was time-dependently suppressed by apoptotic insults including serum deprivation, staurosporine, and C2-ceramide, concomitant with depletion of intracellular ATP and production of reactive oxygen species. Signifying a putative relationship among these events, Ipump was highly sensitive to changes in ATP and reactive oxygen species levels. Moreover, the apoptosis-associated Na+, K+-pump failure and serum deprivation-induced neuronal death were antagonized by pyruvate and succinate in ATP- and reactive-oxygen-species-dependent manners. We suggest that failure of the Na+, K+-pump as a result of a combination of energy deficiency and production of reactive oxygen species is a common event in the apoptotic cascade; preserving the pump activity provides a neuroprotective strategy in certain pathological conditions.
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PMID:Apoptotic insults impair Na+, K+-ATPase activity as a mechanism of neuronal death mediated by concurrent ATP deficiency and oxidant stress. 1267 86


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