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)

The nonstructural protein NS3 of the prototypic flavivirus, yellow fever virus, was investigated for possession of an NTPase activity. The entire NS3 protein coding sequence and an amino-terminal truncated version thereof were engineered into Escherichia coli expression plasmids. Bacteria harboring these plasmids produced the expected polypeptides, which upon cell disruption were found in an insoluble aggregated material considerably enriched for the NS3-related polypeptides. Solubilization and renaturation of these materials, followed by examination of their ability to hydrolyze ATP, revealed an ATPase activity present in both the full-length and amino-terminal truncated NS3 preparations but not in a similarly prepared fraction from E. coli cells engineered to express an unrelated polypeptide. The amino-terminal truncated NS3 polypeptide was further enriched to greater than 95% purity by ion-exchange and affinity chromatography. Throughout the purification scheme, the ATPase activity cochromatographed with the recombinant NS3 polypeptide. The enzymatic activity of the purified material was shown to be a general NTPase and was dramatically stimulated by the presence of particular single-stranded polyribonucleotides. These results are discussed in view of similar activities identified for proteins of other positive-strand RNA viruses.
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PMID:RNA-stimulated NTPase activity associated with yellow fever virus NS3 protein expressed in bacteria. 838 Apr 74

The relationship between the intracellular ATP concentration [ATP](i) and the electrical properties of principal cells was investigated in Malpighian tubules of the yellow fever mosquito, Aedes aegypti. Under control conditions, [ATP](i) was 0.91 mmol l(-1), the input resistance of the principal cell (R(pc)) was 334.1 k Omega, and the basolateral membrane was marked by a large K(+)-conductance and a membrane voltage (V(bl)) of -75.8 mV. Peritubular cyanide (CN, 0.3 mmol l(-1)) reduced [ATP](i) to 0.08 mmol l(-1) in less than 2 min; however, V(bl) dropped to -8 mV and R(pc) increased to 3150.8 k Omega in 8 min, while the K(+)-conductance of the basolateral membrane disappeared. Upon washout of CN, V(bl) and R(pc) returned to control values within 2 min, and the basolateral membrane recovered its K(+)-conductance. The recovery of normal [ATP](i) took 15 min. Dose-dependence and EC(50) values for the CN-inhibition of V(bl) and the increase in R(pc) were strikingly similar (184.0 micromol l(-1) and 164.4 micromol l(-1)). Similar effects of metabolic inhibition were observed with dinitrophenol (DNP), but the EC(50) values were 50.3 micromol l(-1) and 71.7 micromol l(-1) for the effects on V(bl) and R(pc), respectively. Barium, a blocker of K(+)-channels, significantly hyperpolarized V(bl) to -89.1 mV and increased R(pc) to 769.4 k Omega under control conditions, but had no effects during metabolic inhibition. These results illustrate a temporal relationship between [ATP](i) and electrogenic and conductive transport pathways in principal cells that is consistent with the role of ATP as an integrator of transport steps at apical and basolateral membranes of the cell. When [ATP](i) drops to levels that are 10% of control, the V-type H(+)-ATPase is inhibited, preventing the extrusion of K(+) to the tubule lumen. At the same time, basolateral membrane K(+)-channels close, preventing the entry of K(+) from the hemolymph. Intracellular K(+) homeostasis is thus protected during metabolic inhibition, allowing the cell to re-establish K(+) transport when ATP is synthesized again.
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PMID:The dependence of electrical transport pathways in Malpighian tubules on ATP. 1247 94

The V-type H(+)-ATPase is thought to provide the driving force for transepithelial electrolyte and fluid secretion in Malpighian tubules. To confirm the presence of this proton pump in Malpighian tubules of the yellow fever mosquito Aedes aegypti, we used several antibodies raised against the V-type H(+)-ATPase of Manduca sexta. Western blot analysis confirmed the presence of the V-type H(+)-ATPase in Malpighian tubules of Aedes aegypti. In situ immunostaining identified the V-type H(+)-ATPase at the apical membrane of the mitochondrion-rich brush border of principal cells. The V-type H(+)-ATPase was not found in stellate cells. Measurements of ATPase activity revealed that bafilomycin-sensitive and NO(3)(-)-sensitive ATPase activity accounted for 50-60% of total ATPase activity in crude extracts of Malpighian tubules. No significant ouabain- or vanadate-sensitive Na(+)/K(+)-ATPase activity was detected. These results support the conclusion reached previously in electrophysiological studies that the mechanisms for transepithelial electrolyte secretion in the Aedes Malpighian tubules rely on the V-type H(+)-ATPase as the principal energizer of epithelial transport. Measures of transepithelial Na(+) and K(+) secretion and estimates of the H(+) flux mediated by the V-type H(+)-ATPase suggest a 1:1 stoichiometry for Na(+)/H(+) and K(+)/H(+) exchange transport across the apical membrane.
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PMID:The V-type H(+)-ATPase in Malpighian tubules of Aedes aegypti: localization and activity. 1277 Nov 70

Transport across insect epithelia is thought to depend on the activity of a vacuolar-type proton ATPase (V-ATPase) that energizes ion transport through a secondary proton/cation exchanger. Although several of the subunits of the V-ATPase have been cloned, the molecular identity of the exchanger has not been elucidated. Here, we present the identification of sodium/proton exchanger isoform 3 (NHE3) from yellow fever mosquito, Aedes aegypti (AeNHE3). AeNHE3 localizes to the basal plasma membrane of Malpighian tubule, midgut and the ion-transporting sector of gastric caeca. Midgut expression of NHE3 shows a different pattern of enrichment between larval and adult stages, implicating it in the maintenance of regional pH in the midgut during the life cycle. In all tissues examined, NHE3 predominantly localizes to the basal membrane. In addition the limited expression in intracellular vesicles in the median Malpighian tubules may reflect a potential functional versatility of NHE3 in a tissue-specific manner. The localization of V-ATPase and NHE3, and exclusion of Na+/K+-ATPase from the distal ion-transporting sector of caeca, indicate that the role of NHE3 in ion and pH regulation is intricately associated with functions of V-ATPase. The AeNHE3 complements yeast mutants deficient in yeast NHEs, NHA1 and NHX1. To further examine the functional property of AeNHE3, we expressed it in NHE-deficient fibroblast cells. AeNHE3 expressing cells were capable of recovering intracellular pH following an acid load. The recovery was independent of the large cytoplasmic region of AeNHE3, implying this domain to be dispensable for NHE3 ion transport function. 22Na+ uptake studies indicated that AeNHE3 is relatively insensitive to amiloride and EIPA and is capable of Na+ transport in the absence of the cytoplasmic tail. Thus, the core domain containing the transmembrane regions of NHE3 is sufficient for pH recovery and ion transport. The present data facilitate refinement of the prevailing models of insect epithelial transport by incorporating basal amiloride-insensitive NHE3 as a critical mediator of transepithelial ion and fluid transport and likely in the maintenance of intracellular pH.
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PMID:Molecular characterization of sodium/proton exchanger 3 (NHE3) from the yellow fever vector, Aedes aegypti. 1694 93

Infectious diseases caused by flaviviruses are important emerging public health concerns and new vaccines and therapeutics are urgently needed. The NS3 protein from flavivirus is a multifunctional protein with protease, helicase and nucleoside 5' triphosphatase activities (NTPase). Thus, NS3 plays a crucial role in viral replication and represents an interesting target for the development of specific antiviral inhibitors. We have solved the structure of an enzymatically active fragment of the dengue virus NTPase/ helicase C-terminal catalytic domain in several related crystal forms. The structure is composed of three domains, bears an asymmetric distribution of charges and comprises a tunnel large enough to accommodate single strand RNA. A concave face formed by domains 2 and 3 is proposed to bind a nucleic acid duplex substrate. Comparison of the various copies of dengue and yellow fever virus NS3 NTPase/helicase catalytic domains reveals mobile regions of the enzyme. Such dynamic behaviour is likely to be coupled with directional translocation along the single strand nucleic acid substrate during strand separation. We used structure-based site directed mutagenesis to identify regions of the enzyme that are crucial for its ATPase or nucleic acid duplex unwinding activity.
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PMID:Towards the design of flavivirus helicase/NTPase inhibitors: crystallographic and mutagenesis studies of the dengue virus NS3 helicase catalytic domain. 1731 56

The NS3 protein of Japanese encephalitis virus (JEV) is a large multifunctional protein possessing protease, helicase, and nucleoside 5'-triphosphatase (NTPase) activities, and plays important roles in the processing of a viral polyprotein and replication. To clarify the enzymatic properties of NS3 protein from a structural point of view, an enzymatically active fragment of the JEV NTPase/helicase catalytic domain was expressed in bacteria and the crystal structure was determined at 1.8 A resolution. JEV helicase is composed of three domains, displays an asymmetric distribution of charges on its surface, and contains a tunnel large enough to accommodate single-stranded RNA. Each of the motifs I (Walker A motif), II (Walker B motif) and VI was composed of an NTP-binding pocket. Mutation analyses revealed that all of the residues in the Walker A motif (Gly(199), Lys(200) and Thr(201)), in addition to the polar residues within the NTP-binding pocket (Gln(457), Arg(461) and Arg(464)), and also Arg(458) in the outside of the pocket in the motif IV were crucial for ATPase and helicase activities and virus replication. Lys(200) was particularly indispensable, and could not be exchanged for other amino acid residues without sacrificing these activities. The structure of the NTP-binding pocket of JEV is well conserved in dengue virus and yellow fever virus, while different from that of hepatitis C virus. The detailed structural comparison among the viruses of the family Flaviviridae should help in clarifying the molecular mechanism of viral replication and in providing rationale for the development of appropriate therapeutics.
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PMID:Crystal structure of the catalytic domain of Japanese encephalitis virus NS3 helicase/nucleoside triphosphatase at a resolution of 1.8 A. 1820 43

Similar to many flavivirus types including Dengue and yellow fever viruses, the nonstructural NS3 multifunctional protein of West Nile virus (WNV) with an N-terminal serine proteinase domain and an RNA triphosphatase, an NTPase domain, and an RNA helicase in the C-terminal domain is implicated in both polyprotein processing and RNA replication and is therefore a promising drug target. To exhibit its proteolytic activity, NS3 proteinase requires the presence of the cofactor encoded by the upstream NS2B sequence. During our detailed investigation of the biology of the WNV helicase, we characterized the ATPase and RNA/DNA unwinding activities of the full-length NS2B-NS3 proteinase-helicase protein as well as the individual NS3 helicase domain lacking both the NS2B cofactor and the NS3 proteinase sequence and the individual NS3 proteinase-helicase lacking only the NS2B cofactor. We determined that both the NS3 helicase and NS3 proteinase-helicase constructs are capable of unwinding both the DNA and the RNA templates. In contrast, the full-length NS2B-NS3 proteinase-helicase unwinds only the RNA templates, whereas its DNA unwinding activity is severely repressed. Our data suggest that the productive, catalytically competent fold of the NS2B-NS3 proteinase moiety represents an essential component of the RNA-DNA substrate selectivity mechanism in WNV and, possibly, in other flaviviruses. Based on our data, we hypothesize that the mechanism we have identified plays a role yet to be determined in WNV replication occurring both within the virus-induced membrane-bound replication complexes in the host cytoplasm and in the nuclei of infected cells.
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PMID:The two-component NS2B-NS3 proteinase represses DNA unwinding activity of the West Nile virus NS3 helicase. 1844 76

Recently, Na(+)/K(+)-ATPase has been detected in the luminal membrane of the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti) with immunohistochemical techniques. In this study, the possible involvement of this ATPase in strong alkalinization was investigated on the level of whole larvae, isolated and perfused midgut preparations and on the molecular level of the Na(+)/K(+)-ATPase protein. Ouabain (5 mM) did not inhibit the capability of intact larval mosquitoes to alkalinize their anterior midgut. Also in isolated and perfused midgut preparations the perfusion of the lumen with ouabain (5 mM) did not result in a significant change of the transepithelial voltage or the capacity of luminal alkalinization. Na(+)/K(+)-ATPase activity was completely abolished when KCl was substituted with choline chloride, suggesting that the enzyme cannot act as an ATP-driven Na(+)/H(+)-exchanger. Altogether the results of the present investigation indicate that apical Na(+)/K(+)-ATPase is not of direct importance for strong luminal alkalinization in the anterior midgut of larval yellow fever mosquitoes.
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PMID:Strong alkalinization in the anterior midgut of larval yellow fever mosquitoes (Aedes aegypti): involvement of luminal Na+/K+-ATPase. 1904 14

Active transepithelial cation transport in insects was initially discovered in Malpighian tubules, and was subsequently also found in other epithelia such as salivary glands, labial glands, midgut and sensory sensilla. Today it appears to be established that the cation pump is a two-component system of a H(+)-transporting V-ATPase and a cation/nH(+) antiporter. After tracing the discovery of the V-ATPase as the energizer of K(+)/nH(+) antiport in the larval midgut of the tobacco hornworm Manduca sexta we show that research on the tobacco hornworm V-ATPase delivered important findings that emerged to be of general significance for our knowledge of V-ATPases, which are ubiquitous and highly conserved proton pumps. We then discuss the V-ATPase in Malpighian tubules of the fruitfly Drosophila melanogaster where the potential of post-genomic biology has been impressively illustrated. Finally we review an integrated physiological approach in Malpighian tubules of the yellow fever mosquito Aedes aegypti which shows that the V-ATPase delivers the energy for both transcellular and paracellular ion transport.
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PMID:Vacuolar-type proton pumps in insect epithelia. 1944 71

Isolated Malpighian tubules of the yellow fever mosquito secrete NaCl and KCl from the peritubular bath to the tubule lumen via active transport of Na(+) and K(+) by principal cells. Lumen-positive transepithelial voltages are the result. The counter-ion Cl(-) follows passively by electrodiffusion through the paracellular pathway. Water follows by osmosis, but specific routes for water across the epithelium are unknown. Remarkably, the transepithelial secretion of NaCl, KCl and water is driven by a H(+) V-ATPase located in the apical brush border membrane of principal cells and not the canonical Na(+), K(+) -ATPase. A hypothetical cation/H(+) exchanger moves Na(+) and K(+) from the cytoplasm to the tubule lumen. Also remarkable is the dynamic regulation of the paracellular permeability with switch-like speed which mediates in part the post-blood-meal diuresis in mosquitoes. For example, the blood meal the female mosquito takes to nourish her eggs triggers the release of kinin diuretic peptides that (i) increases the Cl(-) conductance of the paracellular pathway and (ii) assembles V(1) and V(0) complexes to activate the H(+) V-ATPase and cation/H(+) exchange close by. Thus, transcellular and paracellular pathways are both stimulated to quickly rid the mosquito of the unwanted salts and water of the blood meal. Stellate cells of the tubule appear to serve a metabolic support role, exporting the HCO(3)(-) generated during stimulated transport activity. Septate junctions define the properties of the paracellular pathway in Malpighian tubules, but the proteins responsible for the permselectivity and barrier functions of the septate junction are unknown.
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PMID:Transcellular and paracellular pathways of transepithelial fluid secretion in Malpighian (renal) tubules of the yellow fever mosquito Aedes aegypti. 2094 39


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