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)

Using Fura-2-loaded human platelets we studied the nature of the mechanisms involved in Ca2+ signalling mediated by H2O2. In a Ca2+-free medium, H2O2 (10 microM-100 mM) induced a concentration-dependent increase in [Ca2+]i. Depletion of either agonist-sensitive or mitochondrial Ca2+ pools reduced this effect while depletion of both stores abolished it. Xestospongin C, an inositol 1,3,5-trisphosphate (IP3) receptor inhibitor, reduced Ca2+ release evoked by 1 mM H2O2 by 45%, indicating that H2O2-induced Ca2+ release involves interaction with IP3 receptors. Blockade of the IP3 turnover by lithium or treatment with U-73122 did not modify H2O2-induced Ca2+ release from the agonist-sensitive pool, suggesting the involvement of a mechanism independent of IP3 generation. H2O2 inhibited Ca2+ reuptake into the agonist-sensitive stores mediated by the sarcoendoplasmic reticulum Ca2+ ATPase (SERCA). Thimerosal (5 microM), a sulphydryl reagent, induced Ca2+ release from the agonist-sensitive stores. This event was impaired by treatment with 2 mM DTT, which also inhibited H2O2-induced Ca2+ release from the agonist-sensitive pool but not from mitochondria. H2O2 reduced the ability of the plasma membrane Ca2+ ATPase (PMCA) to extrude Ca2+ by 75%, an effect that was unaffected by DTT. Consistent with this, thimerosal did not modify the PMCA activity. Finally, exposure to H2O2 triggered platelet aggregation, which was slower than that observed after agonist stimulation. We conclude that H2O2 induced Ca2+ release from agonist-sensitive stores by oxidation of sulphydryl groups in SERCA and the IP3 receptors independently of IP3 generation. In addition, H2O2 induced Ca2+ release from mitochondria and inhibited the PMCA activity by different mechanisms in human platelets.
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PMID:Effect of hydrogen peroxide on Ca2+ mobilisation in human platelets through sulphydryl oxidation dependent and independent mechanisms. 1503 1

In bacteria, the RecA protein plays important roles in a number of DNA recombination and repair processes, including homologous recombination, SOS induction and recombinational DNA repair. We have explored the idea that the Escherichia coli RecA protein's functions could be controlled by small molecules. We investigated the 2:1 complex of zinc(II) with 1,4-dithio-l-threitol (l-DTT) that inhibits the E. coli rho transcription terminator, which is a hexameric ATP motor protein and is structurally homologous to RecA. We found that both the complex and ZnCl(2) inhibit the single-stranded DNA-dependent ATPase activity of RecA at sub-millimolar concentrations. Investigation of a variety of metal dications (0.4 mM final concentration) determined that zinc(II), copper(II) and mercury(II) all induce the precipitation of RecA, while the dichloride salts of calcium, manganese, barium, cobalt, and nickel do not. The inhibition of RecA activity by Zn(II), Cu(II) and Hg(II) results from the metal-dependent initiation of RecA aggregation. These observations may have implications for the design of biophysical experiments requiring solid-phase RecA protein, for a more complete understanding of metal toxicities, and for the design of metal-chelate inhibitors of prokaryotic DNA repair.
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PMID:Inhibition of the Escherichia coli RecA protein: zinc(II), copper(II) and mercury(II) trap RecA as inactive aggregates. 1552 26

We have measured fluid secretion rate in Rhodnius prolixus upper Malpighian tubules (UMT) stimulated to secrete with 5-OH-tryptamine. We used double perfusions in order to have access separately to the basolateral and to the apical cell membranes. Thirteen pharmacological agents were applied: ouabain, Bafilomycin A(1), furosemide, bumetanide, DIOA, Probenecid, SITS, acetazolamide, amiloride, DPC, BaCl(2), pCMBS and DTT. These agents are known to block different ion transport functions, namely ATPases, co- and/or counter-transporters and ion and water channels. The basic assumption is that water movement changes reflect changes in ion transport mechanisms, which we localize as follows: (i) At the basolateral cell membrane, fundamental are a Na(+)-K(+)-2Cl(-) cotransporter and a Cl(-)-HCO(3) (-) exchanger; of intermediate importance are the Na(+)-K(+)-ATPase, Cl(-) channels and Rp-MIP water channels; K(+) channels play a lesser role: (ii) At the apical cell membrane, most important are a K(+)-Cl(-) cotransport that is being located for the first time, a V-H(+)-ATPase; and a Na(+)-H(+) exchanger; a urate-anion exchanger and K(+) channels are less important, while Cl(-) channels are not important at all. A tentative model for the function of the UMT cell is presented.
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PMID:A model for fluid secretion in Rhodnius upper Malpighian tubules (UMT). 1570 74

By screening 1,990 compounds from the National Cancer Institute diversity set library against human topoisomerase IIalpha, we identified a novel catalytic topoisomerase II inhibitor NSC35866, a S6-substituted analogue of thioguanine. In addition to inhibiting the DNA strand passage reaction of human topoisomerase IIalpha, NSC35866 also inhibited its ATPase reaction. NSC35866 primarily inhibited DNA-stimulated ATPase activity, whereas DNA-independent ATPase activity was less sensitive to inhibition. We compared the mode of topoisomerase II ATPase inhibition induced by NSC35866 with that of 12 other substituted purine analogues of different chemical classes. The ability of thiopurines with free SH functionalities to inhibit topoisomerase II ATPase activity was completely abolished by DTT, suggesting that these thiopurines inhibit topoisomerase II ATPase activity by covalently modifying free cysteine residues. In contrast, NSC35866 as well as two O6-substituted guanine analogues, O6-benzylguanine and NU2058, could inhibit topoisomerase II ATPase activity in the presence of DTT, indicating that they have a different mechanism of inhibition. NSC35866 did not increase the level of topoisomerase II covalent cleavable complexes with DNA, indicating that it is a catalytic inhibitor and not a poison. NSC35866 was also capable of inducing a salt-stable complex of topoisomerase II on closed circular DNA. In accordance with these biochemical data, NSC35866 could antagonize etoposide-induced cytotoxicity and DNA breaks in human and murine cancer cells, confirming that NSC35866 also functions as a catalytic topoisomerase II inhibitor in cells.
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PMID:Substituted purine analogues define a novel structural class of catalytic topoisomerase II inhibitors. 1610 1

Nitrovasodilators-sodium nitroprusside (SNP; 10(-9)-10(-4) M) and 3-morpholino-sydnonimine (SIN-1; 10(-9)-10(-4) M) produced concentration-dependent relaxation of the fourth generation sheep pulmonary artery, preconstricted with 5-hydroxytryptamine (1 microM). Oxidizing agents [oxidized glutathione (GSSG, 1 mM) and CuSO4 (5 and 20 microM)] and reducing agents [dithiothreitol (DTT, 0.1 mM), ascorbic acid (1 mM) and reduced glutathione (GSH, 1 mM)] caused opposite effects on nitric oxide (NO)-induced vasodilation in the artery. Ascorbic acid and GSH potentiated the NO responses, while GSSG and CuSO4 inhibited relaxation caused by the nitrovasodilators. DTT, however, reduced the relaxant potency and efficacy of SNP and SIN-1. Pretreatment of the pulmonary artery strips with DTT (0.1 mM) inhibited SNP (10 microM)-induced Na(+)-K(+)-ATPase activity, while ascorbic acid (1 mM) and GSH (1 mM) had no effect either on basal or SNP (10 microM)-stimulated 86Rb uptake, an index of Na(+)-K(+)-ATPase activity, in ovine pulmonary artery. The results suggest that reducing agents like ascorbic acid may have beneficial effect in improving the vascular function under oxidative stress.
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PMID:Effects of oxidizing and reducing agents on ovine pulmonary artery responses to nitric oxide donors, sodium nitroprusside and 3-morpholino-sydnonimine. 1717 68

Donors of nitroxyl (HNO), the reduced congener of nitric oxide (NO), exert positive cardiac inotropy/lusitropy in vivo and in vitro, due in part to their enhancement of Ca(2+) cycling into and out of the sarcoplasmic reticulum. Here we tested whether the cardiac action of HNO further involves changes in myofilament-calcium interaction. Intact rat trabeculae from the right ventricle were mounted between a force transducer and a motor arm, superfused with Krebs-Henseleit (K-H) solution (pH 7.4, room temperature) and loaded iontophoretically with fura-2 to determine [Ca(2+)](i). Sarcomere length was set at 2.2-2.3 microm. HNO donated by Angeli's salt (AS; Na(2)N(2)O(3)) dose-dependently increased both twitch force and [Ca(2+)](i) transients (from 50 to 1000 microm). Force increased more than [Ca(2+)](i) transients, especially at higher doses (332 +/- 33% versus 221 +/- 27%, P < 0.01 at 1000 microm). AS/HNO (250 microm) increased developed force without changing Ca(2+) transients at any given [Ca(2+)](o) (0.5-2.0 mm). During steady-state activation, AS/HNO (250 microm) increased maximal Ca(2+)-activated force (F(max), 106.8 +/- 4.3 versus 86.7 +/- 4.2 mN mm(-2), n = 7-8, P < 0.01) without affecting Ca(2+) required for 50% activation (Ca(50), 0.44 +/- 0.04 versus 0.52 +/- 0.04 microm, not significant) or the Hill coefficient (4.75 +/- 0.67 versus 5.02 +/- 1.1, not significant). AS/HNO did not alter myofibrillar Mg-ATPase activity, supporting an effect on the myofilaments themselves. The thiol reducing agent dithiothreitol (DTT, 5.0 mm) both prevented and reversed HNO action, confirming AS/HNO redox sensitivity. Lastly, NO (from DEA/NO) did not mimic AS/HNO cardiac effects. Thus, in addition to reported changes in Ca(2+) cycling, HNO also acts as a cardiac Ca(2+) sensitizer, augmenting maximal force without altering actomyosin ATPase activity. This is likely to be due to modulation of myofilament proteins that harbour reactive thiolate groups that are targets of HNO.
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PMID:Nitroxyl increases force development in rat cardiac muscle. 1734 60

Plant chloroplasts are particularly threatened by free radical attack. We incubated purified soluble spinach chloroplast F(0)F(1) (CF(0)F(1), EC 3.6.3.34) with an Fe(2+)/H(2)O(2)/ascorbate system, and about 60% inactivation of the ATPase activity was reached after 60 min. Inactivation was not prevented by omission of H(2)O(2), by addition of catalase or superoxide dismutase, nor by the scavengers mannitol, DMSO, or BHT. No evidence for enzyme fragmentation or oligomerization was detected by SDS-PAGE. The chloroplast ATP synthase is resistant to attack by the reactive oxygen species commonly found at the chloroplast level. DTT in the medium completely prevented the inhibition, and its addition after the inhibition partially recovered the activity of the enzyme. CF(0)F(1) thiol residues were lost upon oxidation. The rate of thiol modification was faster than the rate of enzyme inactivation, suggesting that the thiol residues accounting for the inhibition may be hindered. Enzyme previously oxidized by iodobenzoate was not further inhibited by the oxidative system. The production of ascorbyl radical was identified by EPR and is possibly related to CF(0)F(1) inactivation. It is thus suggested that the ascorbyl radical, which accumulates under plant stress, might regulate CF(0)F(1).
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PMID:Inhibition of spinach chloroplast F0F1 by an Fe2+/ascorbate/H2O2 system. 1787 May 88

Myosin can be precipitated from soluble fraction under different assay conditions. This paper describes a new method for precipitating myosin V from rat brain soluble fraction. Brains were homogenized in 50 mM imidazole/HCl buffer, pH 8.0, containing 10 mM EDTA/EGTA, 250 mM sucrose, 1 mM DTT and 1 mM benzamidine, centrifuged at 45000 x g for 40 min and the supernatant was frozen at -20 degrees C. Forty-eight hours later, the supernatant was thawed, centrifuged at 45000 x g for 40 min and the precipitate was washed in 20 mM imidazole buffer pH 8.0. SDS/PAGE analysis showed four polypeptides in the precipitate: 205, 150, 57 and 43 kDa. The precipitate presented high Mg(2+)-ATPase activity, which co-purifies with p205. This polypeptide was recognized by a specific myosin V antibody and was proteolised by calpain, generating two stable polypeptides: p130 and p90. The Mg(2+)-ATPase activity was not stimulated by calcium in both the absence and presence of exogenous calmodulin and the K+/EDTA-ATPase activity represented 25% of the Mg(2+)-ATPase activity. In this work, myosin V from rat brain was precipitated by freezing the soluble fraction and was co-purificated with a 45 kDa polypeptide.
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PMID:A new method to precipitate myosin V from rat brain soluble fraction. 1788 23

Autosomal dominant renal hypomagnesemia, associated with hypocalciurea, has been linked to a G to A mutation at nucleotide position 121 in the FXYD2 gene, resulting in the substitution of Gly with Arg at residue 41 of the protein. FXYD2, also called the Na,K-ATPase gamma-subunit, binds to Na,K-ATPase and influences its cation affinities. In this paper, we provide evidence for the molecular mechanism underlying the dominant character of the disorder. Co-immunoprecipitation experiments using tagged FXYD2 proteins demonstrated that wild type FXYD2 proteins oligomerise. Moreover, FXYD2-G41R also shows oligomerisation with itself and with the wild type protein. In the case of FXYD2-G41R, however, formation of homo-oligomers was prevented by addition of DTT or introduction of the C52A mutation. Finally, we demonstrated that artificial glycosylation of the wild type FXYD2 is reduced when co-expressed with FXYD2-G41R. These data indicate that binding of FXYD2-G41R to wild type FXYD2 subunit might abrogate the routing of wild type FXYD2 to the plasma membrane thus causing the dominant nature of this mutation.
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PMID:Impaired routing of wild type FXYD2 after oligomerisation with FXYD2-G41R might explain the dominant nature of renal hypomagnesemia. 1798 Jun 99

Previously we showed that following hypoxia there is an increase in nuclear Ca(2+)-influx and Ca(2+)/calmodulin-dependent protein kinase IV activity (CaMK IV) in the cerebral cortex of term guinea pig fetus. The present study tests the hypothesis that clonidine administration will prevent hypoxia-induced increased neuronal nuclear Ca(2+)-influx and increased CaMK IV activity, by blocking high-affinity Ca(2+)-ATPase. Studies were conducted in 18 pregnant guinea pigs at term, normoxia (Nx, n=6), hypoxia (Hx, n=6) and clonidine with Hx (Hx+Clo, n=6). The pregnant guinea pig was exposed to a decreased FiO(2) of 0.07 for 60 min. Clonidine, an imidazoline inhibitor of high-affinity Ca(2+)-ATPase, was administered 12.5 microg/kg IP 30 min prior to hypoxia. Hypoxia was determined biochemically by ATP and phosphocreatine (PCr) levels. Nuclei were isolated and ATP-dependent (45)Ca(2+)-influx was determined. CaMK IV activity was determined by (33)P-incorporation into syntide 2 for 2 min at 37 degrees C in a medium containing 50mM HEPES (pH 7.5), 2mM DTT, 40muM syntide 2, 0.2mM (33)P-ATP, 10mM magnesium acetate, 5 microM PKI 5-24, 2 microM PKC 19-36 inhibitor peptides, 1 microM microcystine LR, 200 microM sodium orthovanadate and either 1mM EGTA (for CaMK IV-independent activity) or 0.8mM CaCl(2) and 1mM calmodulin (for total activity). ATP (mumoles/gbrain) values were significantly different in the Nx (4.62+/-0.2), Hx (1.65+/-0.2, p<0.05 vs. Nx), and Hx+Clo (1.92+/-0.6, p<0.05 vs. Nx). PCr (mumoles/g brain) values in the Nx (3.9+/-0.1), Hx (1.10+/-0.3, p<0.05 vs. Nx), and Hx+Clo (1.14+/-0.3, p<0.05 vs. Nx). There was a significant difference between nuclear Ca(2+)-influx (pmoles/mg protein/min) in Nx (3.98+/-0.4), Hx (10.38+/-0.7, p<0.05 vs. Nx), and Hx+Clo (7.35+/-0.9, p<0.05 vs. Nx, p<0.05 vs. Hx), and CaM KIV (pmoles/mg protein/min) in Nx (1314.00+/-195.4), Hx (2315.14+/-148.5, p<0.05 vs. Nx), and Hx+Clo (1686.75+/-154.3, p<0.05 vs. Nx, p<0.05 vs. Hx). We conclude that the mechanism of hypoxia-induced increased nuclear Ca(2+)-influx is mediated by high-affinity Ca(2+)-ATPase and that CaMK IV activity is nuclear Ca(2+)-influx-dependent. We speculate that hypoxia-induced alteration of high-affinity Ca(2+)-ATPase is a key step that triggers nuclear Ca(2+)-influx, leading to CREB protein-mediated increased expression of apoptotic proteins and hypoxic neuronal death.
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PMID:Mechanism of Ca2+-influx and Ca2+/calmodulin-dependent protein kinase IV activity during in utero hypoxia in cerebral cortical neuronal nuclei of the guinea pig fetus at term. 1857 21


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