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)

Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a multifunctional protein known to be involved in the regulation of transcription, translation, nuclear transport, and signal transduction. To systematically obtain insight into mechanisms of hnRNP K activities, we set out to identify protein factors that interact with hnRNP K by using glutathione S-transferase-hnRNP K affinity chromatography followed by liquid chromatography/mass spectrometry/mass spectrometry analysis. Several partner proteins in the K562 cell lysates were identified through this method. One of them is a DEAD box-containing putative RNA helicase, DDX1. In vitro binding and co-immunoprecipitation studies confirmed the protein-protein interaction between hnRNP K with DDX1, and the region spanning amino acids 1-276 of hnRNP K is apparently responsible for its physical interaction with DDX1. Interestingly, their interaction was disrupted by the addition of poly(C), poly(A), and poly(U) RNA substrates. We found that DDX1 was a homopolymeric poly(A) RNA-binding protein. On the other hand, the ATPase activity of the purified recombinant DDX1 protein was stimulated by these homopolymeric RNAs and yeast total RNA but not by DNA. Moreover, the immunoprecipitated DDX1 complex but not purified DDX1 can unwind double-stranded RNA having single-stranded poly(A) overhangs.
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PMID:An RNA helicase, DDX1, interacting with poly(A) RNA and heterogeneous nuclear ribonucleoprotein K. 1218 65

The polarized distribution of Na-K-ATPase at the basolateral membranes of renal tubule epithelial cells is maintained via a tethering interaction with the underlying spectrin-ankyrin cytoskeleton. In this study, we have explored the mechanism underlying the loss of Na-K-ATPase polarity after ischemic injury in Madin-Darby canine kidney (MDCK) cells, utilizing a novel antibody raised against a recently described kidney-specific isoform of ankyrin. In control MDCK cells, ankyrin was colocalized with Na-K-ATPase at the basolateral membrane. ATP depletion resulted in a duration-dependent mislocation of Na-K-ATPase and ankyrin throughout the cytoplasm. Colocalization studies showed a partial overlap between the distribution of ankyrin and Na-K-ATPase at all periods after ATP depletion. By immunoprecipitation with anti-ankyrin antibody, the mislocated Na-K-ATPase remained bound to ankyrin at all time points after ATP depletion. However, the interaction between ankyrin and spectrin was markedly diminished within 3 h of ATP depletion and was completely lost after 6 h. In solution binding assays using a fusion peptide of glutathione S-transferase with the ankyrin binding domain of Na-K-ATPase, a complex with ankyrin was detected at all time points after ATP depletion, but spectrin was lost from the complex in a duration-dependent manner. The loss of spectrin binding was not attributable to spectrin degradation but was associated with hyperphosphorylation of ankyrin. The results suggest that a dissociation of the membrane-cytoskeleton complex at the spectrin-ankyrin interface may contribute to the loss of Na-K-ATPase polarity after ischemic injury and reaffirm a critical adapter role for ankyrin in the normal maintenance of Na-K-ATPase polarity.
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PMID:Dissociation of spectrin-ankyrin complex as a basis for loss of Na-K-ATPase polarity after ischemia. 1240 78

Most plant disease resistance (R) genes known today encode proteins with a central nucleotide binding site (NBS) and a C-terminal Leu-rich repeat (LRR) domain. The NBS contains three ATP/GTP binding motifs known as the kinase-1a or P-loop, kinase-2, and kinase-3a motifs. In this article, we show that the NBS of R proteins forms a functional nucleotide binding pocket. The N-terminal halves of two tomato R proteins, I-2 conferring resistance to Fusarium oxysporum and Mi-1 conferring resistance to root-knot nematodes and potato aphids, were produced as glutathione S-transferase fusions in Escherichia coli. In a filter binding assay, purified I-2 was found to bind ATP rather than other nucleoside triphosphates. ATP binding appeared to be fully dependent on the presence of a divalent cation. A mutant I-2 protein containing a mutation in the P-loop showed a strongly reduced ATP binding capacity. Thin layer chromatography revealed that both I-2 and Mi-1 exerted ATPase activity. Based on the strong conservation of NBS domains in R proteins of the NBS-LRR class, we propose that they all are capable of binding and hydrolyzing ATP.
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PMID:The tomato R gene products I-2 and MI-1 are functional ATP binding proteins with ATPase activity. 1241 11

The plant plasma membrane H(+)-ATPase contains a C-terminal autoinhibitory domain whose displacement from the catalytic site is caused by binding of regulatory 14-3-3 proteins. Members of the highly conserved 14-3-3 family bind their individual target proteins in a sequence-specific and phosphorylation-dependent manner within a central groove, the latter characterized by the presence of highly invariant residues. However, an atypical binding site for 14-3-3s within the H(+)-ATPase has been identified that does not resemble any other 14-3-3 binding motif. Combination of site-directed mutagenesis with glutathione S-transferase pull-down assays points to the importance of the central 14-3-3 groove for the interaction with the apparently unique site of the H(+)-ATPase. Furthermore, a 14-3-3 dimer is essential for binding such unusual motif.
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PMID:Regulatory 14-3-3 proteins bind the atypical motif within the C terminus of the plant plasma membrane H(+)-ATPase via their typical amphipathic groove. 1243 22

The reaction mechanism of the Na,K-ATPase is thought to involve a number of ligand-induced conformational changes. The specific amino acid residues responsible for binding many of the important ligands have been identified; however, details of the specific conformational changes produced by ligand binding are largely undescribed. The experiments described in this paper begin to identify interactions between domains of the Na,K-ATPase alpha-subunit that depend on the presence of particular ligands. The major cytoplasmic loop (between TM4 and TM5), which we have previously shown contains the ATP-binding domain, was overexpressed in bacteria either with a His(6) tag or as a fusion protein with glutathione S-transferase. We have observed that these polypeptides associate in the presence of MgATP. Incubation with [gamma-(32)P]ATP under conditions that result in phosphorylation of the full-length Na,K-ATPase did not result in (32)P incorporation into either the His(6) tag or glutathione S-transferase fusion proteins. The MgATP-induced association was strongly inhibited by prior modification of the fusion proteins with fluorescein isothiocyanate or by simultaneous incubation with 10 microm eosin, indicating that the effect of MgATP is due to interactions within the nucleotide-binding domain. These data are consistent with Na,K-ATPase associating within cells via interactions in the nucleotide-binding domains. Although any functional significance of these associations for ion transport remains unresolved, they may play a role in cell function and in modulating interactions between the Na,K-ATPase and other proteins.
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PMID:Interactions between Na,K-ATPase alpha-subunit ATP-binding domains. 1251 76

Intercalated and inner medullary collecting duct (IMCD) cells of the kidney mediate the transport of H+ by a plasma membrane H+-ATPase. The rate of H+ transport in these cells is regulated by exocytic insertion of H+-ATPase-laden vesicles into the apical membrane. We have shown that the exocytic insertion of proton pumps (H+-ATPase) into the apical membrane of rat IMCD cells, in culture, involves SNARE proteins (syntaxin (synt), SNAP-23, and VAMP). The membrane fusion complex observed in IMCD cells with the induction of proton pump exocytosis not only included these SNAREs but also the H+-ATPase. Based on these observations, we suggested that the targeting of these vesicles to the apical membrane is mediated by an interaction between the H+-ATPase and a specific t-SNARE. To evaluate this hypothesis, we utilized a "pull-down" assay in which we identified, by Western analysis, the proteins in a rat kidney medullary homogenate that complexed with glutathione S-transferase (GST) fusion syntaxin isoforms attached to Sepharose 4B-glutathione beads. The syntaxin isoforms employed were 1A, 1B, 2, 4, 5, and also 1A that was truncated to exclude the H3 SNARE binding domain (synt-1ADeltaH3). All full-length syntaxin isoforms formed complexes with SNAP-23 and VAMP. Neither GST nor synt-1ADeltaH3 formed complexes with these SNAREs. H+-ATPase (subunits E, a, and c) bound to syntaxin-1A and to a lesser extent to synt-1B but not to synt-1ADeltaH3 or synt-2, -4, and -5. In cultured IMCD cells transfected to express syntaxin truncated for the membrane binding domain (synt-DeltaC), expression of synt-1ADeltaC, but not synt-4DeltaC, inhibited H+-ATPase exocytosis. In conclusion, because all full-length syntaxins examined bound VAMP-2 and SNAP-23, but only non-H3-truncated syntaxin-1 bound H+-ATPase, and synt-1ADeltaC expression by intact IMCD cells inhibited H+-ATPase exocytosis, it is likely that the H+-ATPase binds directly to the H3 domain of syntaxin-1 and not through VAMP-2 or SNAP-23. Interaction between the syntaxin-1A and H+-ATPase is important in the targeted exocytosis of the proton pump to the apical membrane of intercalated cells.
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PMID:Syntaxin isoform specificity in the regulation of renal H+-ATPase exocytosis. 1265 53

Curcumin, a yellow pigment of turmeric (Curcuma longa), is a commonly used spice and a coloring agent in foods, drugs, and cosmetics. Curcumin is known to possess chemopreventive properties in various animal tumor models. In the present study the effect of curcumin on the development of altered hepatic foci (AHF), by using a medium term liver bioassay, has been evaluated. AHF were analyzed by quantitative stereology using the Leica Qwin Image Analysis system from frozen liver sections stained for g-glutamyl transferase, adenosine triphosphatase, glucose-6-phosphatase, alkaline phosphatase, and placental isozyme of glutathione S-transferase. A significant protection on diethylnitrosamine (DEN) initiated and 2-acetylaminofluorene (AAF) promoted AHF by curcumin was observed on these biological markers. The curcumin administration was found to restore the normal levels of the enzymes glutathione S-transferase and g-glutamyl transferase in rat liver following DEN-AAF exposure. Similarly, a significant protection was provided by curcumin in the enzyme-deficient foci for the adenosine triphosphatase-, alkaline phosphatase-, and glucose-6-phosphatase-treated groups in comparison to the DEN-AAF-treated group. These results show that curcumin can effectively suppress the DEN-induced development of AHF in rat liver.
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PMID:Suppression of altered hepatic foci development by curcumin in wistar rats. 1279 5

Polyubiquitination is required for retrotranslocation of proteins from the endoplasmic reticulum back into the cytosol, where they are degraded by the proteasome. We have tested whether the release of a polypeptide chain into the cytosol is caused by a ratcheting mechanism in which the attachment of polyubiquitin prevents the chain from moving back into the endoplasmic reticulum. Using a permeabilized cell system in which major histocompatibility complex class I heavy chains are retrotranslocated under the influence of the human cytomegalovirus protein US11, we demonstrate that polyubiquitination alone is insufficient to provide the driving force for retrotranslocation. Substrate release into the cytosol requires an additional ATP-dependent step. Release requires a lysine 48 linkage of ubiquitin chains. It does not occur when polyubiquitination of the substrate is carried out with glutathione S-transferase (GST)-ubiquitin, and this correlates with poly-GST-ubiquitin not being recognized by a ubiquitin-binding domain in the Ufd1-Npl4 cofactor of the ATPase p97. These data suggest that polyubiquitin does not serve as a ratcheting molecule. Rather, it may serve as a recognition signal for the p97-Ufd1-Npl4 complex, a component implicated in the movement of substrate into the cytosol.
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PMID:Polyubiquitin serves as a recognition signal, rather than a ratcheting molecule, during retrotranslocation of proteins across the endoplasmic reticulum membrane. 1281 30

Alix (ALG-2-interacting protein X) is a 95-kDa protein that interacts with an EF-hand type Ca(2+)-binding protein, ALG-2 (apoptosis-linked gene 2), through its C-terminal proline-rich region. In this study, we searched for proteins that interact with human AlixDeltaC (a truncated form not containing the C-terminal region) by using a yeast two-hybrid screen, and we identified two similar human proteins, CHMP4a and CHMP4b (chromatin-modifying protein; charged multivesicular body protein), as novel binding partners of Alix. The interaction of Alix with CHMP4b was confirmed by a glutathione S-transferase pull-down assay and by co-immunoprecipitation experiments. Fluorescence microscopic analysis revealed that CHMP4b transiently expressed in HeLa cells mainly exhibited a punctate distribution in the perinuclear area and co-localized with co-expressed Alix. The distribution of CHMP4b partly overlapped the distributions of early and late endosomal marker proteins, EEA1 (early endosome antigen 1) and Lamp-1 (lysosomal membrane protein-1), respectively. Transient overexpression of CHMP4b induced the accumulation of ubiquitinated proteins as punctate patterns that were partly overlapped with the distribution of CHMP4b and inhibited the disappearance of endocytosed epidermal growth factor. In contrast, stably expressed CHMP4b in HEK293 cells was observed diffusely in the cytoplasm. Transient overexpression of AlixDeltaC in stably CHMP4b-expressing cells, however, induced formation of vesicle-like structures in which CHMP4b and AlixDeltaC were co-localized. SKD1(E235Q), a dominant negative form of the AAA type ATPase SKD1 that plays critical roles in the endocytic pathway, was co-immunoprecipitated with CHMP4b. Furthermore, CHMP4b co-localized with SKD1(E235Q) as punctate patterns in the perinuclear area, and Alix was induced to exhibit dot-like distributions overlapped with SKD1(E235Q) in HeLa cells. These results suggest that CHMP4b and Alix participate in formation of multivesicular bodies by cooperating with SKD1.
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PMID:The ALG-2-interacting protein Alix associates with CHMP4b, a human homologue of yeast Snf7 that is involved in multivesicular body sorting. 1286 Sep 94

The study investigates the effect of aqueous extract of fenugreek seeds (Trigonella foenum graecum) on lipid peroxidation and antioxidant status in experimental ethanol toxicity in rats. The ability of the seed extract to prevent iron-induced lipid peroxidation in vitro was also investigated. Ethanol feeding for 60 days resulted in significant increases in the activities of serum aspartate transaminase, alanine transaminase and alkaline phosphatase. The levels of serum lipid hydroperoxides and thiobarbituric acid reactive substances in liver and brain were also significantly elevated. Significantly lower activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase and glutathione reductase were observed in liver and brain accompanied by depletion in glutathione, ascorbic acid and alpha-tocopherol concentrations. Activity of Ca(2+) ATPase in brain was significantly lowered. Simultaneous administration of aqueous extract of fenugreek seeds with ethanol prevented the enzymatic leakage and the rise in lipid peroxidation and enhanced the antioxidant potential. The seeds exhibited appreciable antioxidant property in vitro which was comparable with that of reduced glutathione and alpha-tocopherol. Further, histopathological examination of liver and brain revealed that, aqueous extract of fenugreek seeds could offer a significant protection against ethanol toxicity.
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PMID:Protective effect of fenugreek (Trigonella foenum graecum) seeds in experimental ethanol toxicity. 1291 70


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