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)

Na+, K+-ATPase beta2 subunit (NKA1b2) is not only a regulator of Na+, K+-ATPase, but also functions in the interaction between neuron and glia cells as a Ca2+-dependent adhesion molecule. To further study the function of NKA1b2, the anti-NKA1b2 polyclonal antibody was prepared to recognize the outer-membrane carboxyl portion segment of NKA1b2. The coding region for amino acids 190-290 at the carboxyl portion of NKA1b2 (NKA1b2-CP) was sub-cloned into the vector pGEX-4T-2 and introduced into the Escherichia coli BL21(DE3) cell for efficient soluble expression. The amino acid sequence of expressed protein was determined using mass spectrometry following Mascot analysis. After purification, GST-NKA-beta2-CP was used to immunize the adult rabbits following standard protocols. The produced antiserum could detect the NKA1b2 protein expressed not only in the prokaryotic cells (E. coli) but also in the eukaryotic cells (COS7) transfected with NKA1b2 expression vector (pEGFP-NKA1b2). Furthermore, the antiserum was used for determining the localization of NKA1b2 in primary culture of neonatal rat neurons using immunohistochemical technique. Results demonstrated that NKA1b2 was localized both in the cytoplasm and cellular membrane. The preparation of anti-NKA-beta2-CP polyclonal antibody will facilitate further functional study on NKA1b2.
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PMID:Prokaryotic expression, polyclonal antibody preparation, and sub-cellular localization analysis of Na+, K+-ATPase beta2 subunit. 1529 80

Diallyl sulphide (DAS) is a sulphur-containing volatile compound present in garlic (Allium sativum). It has been shown to inhibit a number of chemically induced forms of cancer in experimental animals. The present study demonstrates the inhibitory effect of DAS on the development of diethylnitrosamine (DEN) initiated and 2-acetyl-aminofluorene (2-AAF) promoted preneoplastic altered hepatic foci (AHF) in Wistar rats. AHF were scored and analysed by quantitative stereology using the Image Analysis system from frozen liver sections stained for biological markers, namely glutathione S-transferase, placental form (GST-P), gamma-glutamyl transpeptidase (GGT), adenosine triphosphatase (ATPase), glucose-6-phosphatase (G6 Pase) and alkaline phosphatase (AlkPase). DAS-supplemented rats were found to restore the near-normal levels of enzymes GST-P and GGT when exposed to DEN and 2-AAF. DAS administration following DEN and 2-AAF exposure led to the restoration of enzymic activity of ATPase, G6 Pase and AlkPase, as evident by number and area of the foci. These findings suggest the protective role of DAS in rat hepatocarcinogenesis, by suppressing DEN- and 2-AAF-induced AHF development.
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PMID:Modulation of altered hepatic foci induction by diallyl sulphide in Wistar rats. 1555 53

Indole-3-carbinol (I3C) is a cleavage product of glucobrassicanin, a natural compound present in a wide variety of plant food substances including members of the family Cruciferae. I3C is known to possess cancer-chemopreventive potential in various animal models. The present study reveals the protective effect of I3C on the development of diethylnitrosamine (DEN)-initiated and 2-acetylaminofluorene (AAF)-promoted preneoplastic, altered hepatic foci (AHF) in Wistar rats. I3C was given at dose levels of 0.5 and 1 mg/kg body weight for five consecutive days along with DEN and AAF. AHF were scored and analyzed by quantitative stereology using the Image Analysis System from frozen liver sections stained for positive and negative biological markers of AHF, that is, glutathione S-transferase (GST-P), gamma-glutamyl transpeptidase (GGT), glucose-6-phosphatase (G6Pase), adenosine triphosphatase (ATPase), and alkaline phosphatase (AlkPase). Results revealed the chemopreventive effect of I3C on the DEN-initiated AHF in Wistar rats. The expression of G6Pase, ATPase, and AlkPase was restored in the I3C-supplemented animal. Similarly the induced expression GST-P and GGT also decreased in the animals with I3C administration. The recovery of altered levels of these biomarkers was of comparatively higher magnitude in the animals given a higher dose of I3C (1 mg/kg body weight) in comparison with the animals given 0.5 mg/kg body weight dose of I3C, although no dose-dependence pattern was recorded in I3C-supplemented groups. These results thus suggest the chemopreventive effect of I3C in rat hepatocarcinogenesis by suppressing DEN- and AAF-induced AHF development.
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PMID:Chemopreventive effect of indole-3-carbinol on induction of preneoplastic altered hepatic foci. 1562 69

Vacuolar H(+)-ATPases (V-ATPases) are multi-subunit membrane proteins that couple ATP hydrolysis to the extrusion of protons from the cytoplasm. Although they share a common macromolecular architecture and rotational mechanism with the F(1)F(0)-ATPases, the organization of many of the specialized V-ATPase subunits within this rotary molecular motor remains uncertain. In this study, we have identified sequence segments involved in linking putative stator subunits in the Saccharomyces V-ATPase. Precipitation assays revealed that subunits Vma5p (subunit C) and Vma10p (subunit G), expressed as glutathione-S-transferase fusion proteins in E. coli, are both able to interact strongly with Vma4p (subunit E) expressed in a cell-free system. GST-Vma10p also associated with Vma2p and Vma1p, the core subunits of the ATP-hydrolyzing domain, and was able to self-associate to form a dimer. Mutations within the first 19-residue region of Vma4p, which disrupted interaction with Vma5p in vitro, also prevented the Vma4p polypeptide from restoring V-ATPase function in a complementation assay in vivo. These mutations did not prevent assembly of Vma5p (subunit C) and Vma2p (subunit B) into an inactive complex at the vacuolar membrane, indicating that Vma5p must make multiple interactions involving other V-ATPase subunits. A second, highly conserved region of Vma4p between residues 19 and 38 is involved in binding Vma10p. This region is highly enriched in charged residues, suggesting a role for electrostatic effects in Vma4p-Vma10p interaction. These protein interaction studies show that the N-terminal region of Vma4p is a key factor not only in the stator structure of the V-ATPase rotary molecular motor, but also in mediating interactions with putative regulatory subunits.
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PMID:Defined sites of interaction between subunits E (Vma4p), C (Vma5p), and G (Vma10p) within the stator structure of the vacuolar H+-ATPase. 1575 69

We have shown that the caveolar Na/K-ATPase transmits ouabain signals via multiple signalplexes. To obtain the information on the composition of such complexes, we separated the Na/K-ATPase from the outer medulla of rat kidney into two different fractions by detergent treatment and density gradient centrifugation. Analysis of the light fraction indicated that both PLC-gamma1 and IP3 receptors (isoforms 2 and 3, IP3R2 and IP3R3) were coenriched with the Na/K-ATPase, caveolin-1 and Src. GST pulldown assays revealed that the central loop of the Na/K-ATPase alpha1 subunit interacts with PLC-gamma1, whereas the N-terminus binds IP3R2 and IP3R3, suggesting that the signaling Na/K-ATPase may tether PLC-gamma1 and IP3 receptors together to form a Ca(2+)-regulatory complex. This notion is supported by the following findings. First, both PLC-gamma1 and IP3R2 coimmunoprecipitated with the Na/K-ATPase and ouabain increased this interaction in a dose- and time-dependent manner in LLC-PK1 cells. Depletion of cholesterol abolished the effects of ouabain on this interaction. Second, ouabain induced phosphorylation of PLC-gamma1 at Tyr(783) and activated PLC-gamma1 in a Src-dependent manner, resulting in increased hydrolysis of PIP2. It also stimulated Src-dependent tyrosine phosphorylation of the IP3R2. Finally, ouabain induced Ca(2+) release from the intracellular stores via the activation of IP3 receptors in LLC-PK1 cells. This effect required the ouabain-induced activation of PLC-gamma1. Inhibition of Src or depletion of cholesterol also abolished the effect of ouabain on intracellular Ca(2+).
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PMID:Na/K-ATPase tethers phospholipase C and IP3 receptor into a calcium-regulatory complex. 1597 99

ALG-2 (apoptosis-linked gene 2) is a Ca2+-binding protein that belongs to the PEF (penta-EF-hand) protein family. Alix (ALG-2-interacting protein X)/AIP1 (ALG-2-interacting protein 1), one of its binding partners, interacts with TSG101 and CHMP4 (charged multivesicular body protein 4), which are components of ESCRT-I (endosomal sorting complex required for transport I) and ESCRT-III respectively. In the present study, we investigated the association between ALG-2 and ESCRT-I. By a GST (glutathione S-transferase) pull-down assay using HEK-293T (human embryonic kidney 293T) cell lysates, endogenous TSG101 and two other exogenously expressed ESCRT-I components [hVps28 (human vacuolar protein sorting 28) and hVps37A] were shown to associate with GST-ALG-2 in the presence of Ca2+. By the yeast two-hybrid assay, however, a positive interaction was observed with only TSG101 among the three ESCRT-I components, suggesting that ALG-2 associates with hVps28 and hVps37A indirectly through TSG101. Using various deletion mutants of TSG101, the central PRR (proline-rich region) was found to be sufficient for interaction with ALG-2 by the GST-pull-down assay. Direct binding of ALG-2 to the TSG101 PRR was demonstrated by an overlay assay using biotin-labelled ALG-2 as a probe. In immunofluorescence microscopic analysis of HeLa cells that overexpressed a GFP (green fluorescent protein)-fused ATPase-defective dominant-negative form of SKD1/Vps4B (GFP-SKD1(E235Q)), ALG-2 exhibited a punctate distribution at the perinuclear area and co-localized with GFP-SKD1(E235Q) to aberrant endosomes. This punctate distribution of ALG-2 was markedly diminished by treatment of HeLa cells with a membrane-permeant Ca2+ chelator. Moreover, a Ca2+-binding-defective mutant of ALG-2 did not co-localize with GFP-SKD1(E235Q). Our findings suggest that ALG-2 may function as a Ca2+-dependent accessory protein of the endosomal sorting machinery by interacting directly with TSG101 as well as with Alix.
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PMID:The penta-EF-hand protein ALG-2 interacts directly with the ESCRT-I component TSG101, and Ca2+-dependently co-localizes to aberrant endosomes with dominant-negative AAA ATPase SKD1/Vps4B. 1600 3

A capillary electrophoresis (CE)-based method for the in vitro detection and monitoring of nucleotide-triphosphatase activity is described. This robust and reproducible method was used to investigate GTPase activity of a recombinant protein construct containing the catalytic domain of Human SEPT4/Bradeion beta (GST-rDGTPase). This example application demonstrates that the CE technique can replace classical radioactive methods for GTPase activity assays and may be used as a routine analytical tool. Enzyme kinetics of GST-rDGTPase was studied and yielded the following kinetic parameters: v(max) = 1.7 microM min(-1) +/- 0.1, Km = 1.0 mM +/- 0.3, and apKcat = 9 x 10(-3) s(-1). In addition the effect of co-factors such as Mg2+ and Mn2+ on the catalytic activity was investigated. The described analytical method was also shown to be useful to analyze diphosphated and triphosphated forms of other nucleotides.
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PMID:In vitro monitoring of GTPase activity and enzyme kinetics studies using capillary electrophoresis. 1604 3

The relationship between the expression level of putative drug resistance factors and sensitivity to anticancer drugs in human normal renal proximal tubule epithelial cells (RPTEC) and 3 kinds of renal cell carcinoma (RCC) cells, VMRC-RCW (RCW), OS-RC-2 (OS2), TUHR14TKB (14TKB), was examined. RPTEC exhibited high expression of P-glycoprotein (Pgp), gamma-glutamyl cysteine synthetase (gammaGCS) and cis-diamminedichloroplatinum (II) (CDDP) resistance-related gene 9 (CRR9), low expression of vacuolar ATPase (V-ATPase) and no expression of multidrug resistance-associated protein 1 (MRP1). 14TKB exhibited high expression of gammaGCS and CRR9, low expression of Pgp and V-ATPase, and no expression of MRP1. OS2 showed high expression of CRR9, low expression of Pgp, gammaGCS and MRP1, and no expression of V-ATPase. RCW exhibited high expression of Pgp, MRP1 and CRR9 and low expression of gammaGCS and V-ATPase. The level of expression of the resistance factors varied among the cells. GST activity and GST-pi expression level of each cell were correlated, and there were high levels in OS2 and RPTEC. When the cytotoxicity of anticancer drugs against each cell was measured at 96 h, the sensitivity to CDDP and Doxorubicin (DXR) in RPTEC and RCW was lower than that in the other cells. Sensitivity to DXR was enhanced by treatment with the Pgp inhibitor, Verapamil, in proportion to the Pgp expression level, and the sensitivity to CDDP was increased by the gammaGCS inhibitor, Buthionine sulfoximine, in proportion to the gammaGCS expression level (corresponding to GSH content). Although a significant increase in sensitivity to CDDP was not observed by treatment of RCC with the V-ATPase inhibitor, Bafilomycin, the sensitivity to DXR in Bafilomycin-treated cells increased about 2-fold. However, no relation between drug sensitivity and V-ATPase expression was observed. The features (such as degree of resistance) varied among the RCC cell lines manifesting many resistance factors or to the contrary, lacking or having lowered resistance factors in comparison with normal cells. Therefore, it is necessary in clinical cancer chemotherapy to determine and measure the level of expression of each resistance factor in respective tumor tissue.
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PMID:Relationship between expression of drug-resistance factors and drug sensitivity in normal human renal proximal tubular epithelial cells in comparison with renal cell carcinoma. 1607 62

The transfer RNA gene downstream from the HMR locus in S. cerevisiae functions as part of a boundary (barrier) element that restricts the spread of heterochromatic gene silencing into the downstream region of chromosome III. A genetic screen for identifying additional genes that, when mutated, allow inappropriate spreading of silencing from HMR through the tRNA gene was performed. YTA7, a gene containing bromodomain and ATPase homologies, was identified multiple times. Previously, others had shown that the bromodomain protein Bdf1p functions to restrict silencing at yeast euchromatin-heterochromatin boundaries; therefore we deleted nonessential bromodomain-containing genes to test their effects on heterochromatin spreading. Deletion of RSC2, coding for a component of the RSC chromatin-remodeling complex, resulted in a significant spread of silencing at HMR. Since the bromodomain of YTA7 lacks a key tyrosine residue shown to be important for acetyllysine binding in other bromodomains, we confirmed that a GST-Yta7p bromodomain fusion was capable of binding to histones in vitro. Epistasis analysis suggests that YTA7 and the HMR-tRNA function independently to restrict the spread of silencing, while RSC2 may function through the tRNA element. Our results suggest that multiple bromodomain proteins are involved in restricting the propagation of heterochromatin at HMR.
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PMID:Multiple bromodomain genes are involved in restricting the spread of heterochromatic silencing at the Saccharomyces cerevisiae HMR-tRNA boundary. 1607 23

Osteoclasts are cells specialized for bone resorption. For osteoclast activation, tumor necrosis factor receptor-associated factor 6 (TRAF6) plays a pivotal role. To find new molecules that bind TRAF6 and have a function in osteoclast activation, we employed a proteomic approach. TRAF6-binding proteins were purified from osteoclast cell lysates by affinity chromatography and their identity was disclosed by MS. The identified proteins included several heat shock proteins, actin and actin-binding proteins, and vacuolar ATPase (V-ATPase). V-ATPase, documented for a great increase in expression during osteoclast differentiation, is an important enzyme for osteoclast function; it transports proton to resorption lacunae for hydroxyapatite dissolution. The binding of V-ATPase with TRAF6 was confirmed both in vitro by GST pull-down assays and in osteoclasts by co-immunoprecipitation and confocal microscopy experiments. In addition, the V-ATPase activity associated with TRAF6 increased in osteoclasts stimulated with receptor activator of nuclear factor kappaB ligand (RANKL). Furthermore, a dominant-negative form of TRAF6 abrogated the RANKL stimulation of V-ATPase activity. Our study identified V-ATPase as a TRAF6-binding protein using a proteomics strategy and proved a direct link between these two important molecules for osteoclast function.
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PMID:Proteomic identification of the TRAF6 regulation of vacuolar ATPase for osteoclast function. 1619 1


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