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)

Endocytic vesicles that are involved in the vasopressin-stimulated recycling of water channels to and from the apical membrane of kidney collecting duct principal cells were isolated from rat renal papilla by differential and Percoll density gradient centrifugation. Fluorescence quenching measurements showed that the isolated vesicles maintained a high, HgCl2-sensitive water permeability, consistent with the presence of vasopressin-sensitive water channels. They did not, however, exhibit ATP-dependent luminal acidification, nor any N-ethylmaleimide-sensitive ATPase activity, properties that are characteristic of most acidic endosomal compartments. Western blotting with specific antibodies showed that the 31- and 70-kD cytoplasmically oriented subunits of the vacuolar proton pump were not detectable in these apical endosomes from the papilla, whereas they were present in endosomes prepared in parallel from the cortex. In contrast, the 56-kD subunit of the proton pump was abundant in papillary endosomes, and was localized at the apical pole of principal cells by immunocytochemistry. Finally, an antibody that recognizes the 16-kD transmembrane subunit of oat tonoplast ATPase cross-reacted with a distinct 16-kD band in cortical endosomes, but no 16-kD band was detectable in endosomes from the papilla. This antibody also recognized a 16-kD band in affinity-purified H+ ATPase preparations from bovine kidney medulla. Therefore, early endosomes derived from the apical plasma membrane of collecting duct principal cells fail to acidify because they lack functionally important subunits of a vacuolar-type proton pumping ATPase, including the 16-kD transmembrane domain that serves as the proton-conducting channel, and the 70-kD cytoplasmic subunit that contains the ATPase catalytic site. This specialized, non-acidic early endosomal compartment appears to be involved primarily in the hormonally induced recycling of water channels to and from the apical plasma membrane of vasopressin-sensitive cells in the kidney collecting duct.
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PMID:Apical endosomes isolated from kidney collecting duct principal cells lack subunits of the proton pumping ATPase. 138 76

A monoclonal antibody to vacuolar H+ATPase isolated from bovine kidney medulla was produced and characterized by immunoprecipitation and immunocytochemistry. The antibody, immobilized on beads, specifically immunoprecipitated both solubilized N-ethylmaleimide-sensitive ATPase activity and proton-transporting vesicles from renal microsomes; control experiments with an "irrelevant" monoclonal antibody showed no immunoprecipitated activity. By fluorescent immunocytochemistry, the antibody stained the membranes of intracellular vacuolar compartments in LLC-PK1 cells. Immunocytochemical staining showed that the monoclonal antibody colocalized partially with N-(3-[2,4-dinitrophenyl)amino)propyl)-N-(3-amino-propyl)methylamine, a probe for acidic compartments, with the endocytic markers dextran and transferrin, with the lysosomal probe alpha 2-macroglobulin, and with clathrin. The anti-vacuolar H+ATPase antibody showed no colocalization with staining for mitochondrial H+ATPase. The anti-vacuolar H+ATPase antibody should serve as a specific probe for examining the distribution and dynamics of the vacuolar proton pump in renal epithelial cells.
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PMID:Production and characterization of a monoclonal antibody to vacuolar H+ATPase of renal epithelia. 289 Jun 33

Earlier studies have shown that in Dictyostelium discoideum, a buoyant membrane fraction contained approximately 90% of the vacuolar proton pump (V-H(+)-ATPase) activity, leading to its designation acidosomes. It was proposed that acidosomes may be involved in endocytosis, specially in the acidification of endosomes. In this study we further investigated the putative function(s) of acidosomes. The findings suggest that acidosomes contain abundant receptors for cyclic AMP (CAR1) and that it may be the site for recycling of internalized receptors. Acidosomes also contain an abundance of Rab4 (Bush et al. 1994), a marker for early endosomes. By these criteria, we suggest that the acidosomes are analogous to early or recycling endosome present in mammalian cells. These findings suggest that the structure earlier defined biochemically, morphologically and immunologically as acidosomes may represent early and/or recycling endosomes in this protist.
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PMID:Localization of cyclic-AMP receptors with acidosomes in Dictyostelium discoideum. 762 37

Calcium uptake by microsomal membranes from the cellular slime mould Dictyostelium discoideum was measured using Calcium Green-2 as a fluorescent probe of external free Ca2+ concentration. High-affinity Ca2+ uptake was found to be completely inhibited by low concentrations of vanadate, but not by thapsigargin, suggesting that the activity is mediated by a Ca(2+)-ATPase distinct from sarco(endo)plasmic reticulum type of higher animal cells. On sucrose density gradients, Ca2+ uptake distributes with vacuolar proton pump activity and part of the observed Ca2+ uptake is dependent on the pH gradient generated by the vacuolar-type H(+)-ATPase, indicating that the Ca2+ pump is located on both acidic and non-acidic vesicles, possibly derived from the H(+)-ATPase-rich contractile vacuole complex.
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PMID:Characterisation of an intracellular Ca2+ pump in Dictyostelium. 771 44

Earlier studies have shown that, in Dictyostelium discoideum, approximately 90% of the vacuolar proton pump (V-H(+)-ATPase) activity is present in a buoyant membrane fraction called "acidosomes." In the presence of Mg2+, acidosomes and endocytic vacuoles copurified on equilibrium sucrose gradients, suggesting their reversible association. The association depended on Mg2+ and cytosolic proteins (H. Padh et al., 1991, J. Biol. Chem. 266, 5514-5520, 12123-12126). To further characterize the putative association of acidosomes and endocytic vacuoles, cells were fed dextran-coated superparamagnetic iron colloid plus FITC-dextran to load and label their endocytic vacuoles. The endocytic vesicles were then purified approximately 20-fold at > 60% yield by their retention on a column of fine steel wire in an electromagnetic field in the absence of Mg2+. The fraction retained on a magnet column contained only about 5% of total cellular V-H(+)-ATPase and traces of other organelle markers. In the presence of 1.5 mM Mg2+, however, the retention of V-H(+)-ATPase as well as FITC-dextran was approximately 60% with only traces of contaminant markers. When such preparations were washed with buffer lacking Mg2+ while still in the magnetic field, the endocytic marker (FITC-dextran) remained on the column while V-H(+)-ATPase was eluted selectively. The elute was shown by negative-stain electron microscopy to contain purified acidosomes (saccular membranes studded with V-H(+)-ATPase). The parent material, recovered from the column in the presence of Mg2+, was rich in endocytic vacuoles bearing colloidal iron. In an electron microscope, the endocytic vacuoles were often seen associated with pump-studded acidosomes. The results independently support and extend earlier observation that acidosomes and endocytic vacuoles physically associate in a Mg(2+)-dependent manner. In addition, the procedure provides a rapid method of purifying acidosomes.
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PMID:Electromagnetic purification of endocytic vacuoles and acidosomes from Dictyostelium. 784 Jun 77

To examine the relationship between endosome acidification and receptor trafficking, transferrin receptor trafficking was characterized in Chinese hamster ovary cells in which endosome acidification was blocked by treatment with the specific inhibitor of the vacuolar H(+)-ATPase, bafilomycin A1. Elevating endosome pH slowed the receptor externalization rate to approximately one-half of control but did not affect receptor internalization kinetics. The slowed receptor externalization required the receptor's cytoplasmic domain and was largely eliminated by substitutions replacing either of two aromatic amino acids within the receptor's cytoplasmic YTRF internalization motif. These results confirm, using a specific inhibitor of the vacuolar proton pump, that proper endosome acidification is necessary to maintain rapid recycling of intracellular receptors back to the plasma membrane. Moreover, receptor return to the plasma membrane is slowed in the absence of proper endosome acidification by a signal-dependent mechanism involving the receptor's cytoplasmic tyrosine-containing internalization motif. These results, in conjunction with results from other studies, suggest that the mechanism for clustering receptors in plasma membrane clathrin-coated pits may be an example of a more general mechanism that determines the dynamic distribution of membrane proteins among various compartments with luminal acidification playing a crucial role in this process.
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PMID:Endosome acidification and receptor trafficking: bafilomycin A1 slows receptor externalization by a mechanism involving the receptor's internalization motif. 816 8

Antisera were generated in rabbits against the vacuolar proton pump (V-H(+)-ATPase) purified from Dictyostelium discoideum. The antisera inhibited V-H(+)-ATPase but not F1-ATPase activity and immunoprecipitated and immunoblotted only the polypeptide subunits of the V-H(+)-ATPase from cell homogenates. Immunocytochemical analysis of intact cells and subcellular fractions showed that the predominant immunoreactive organelles were clusters of empty, irregular vacuoles of various sizes and shapes, which corresponded to the acidosomes. The cytoplasmic surfaces of lysosomes, phagosomes and the tubular spongiome of the contractile vacuole also bore the pump antigen. The lumina of multivesicular bodies were often stained intensely; the internalized antigen may have been derived from acidosomes by autophagy. Antibodies against V-H(+)-ATPases from plant and animal cells cross-reacted with the proton pumps of Dictyostelium. Antisera directed against the V-H(+)-ATPase of Dictyostelium decorated a profusion of small vacuoles scattered throughout the cytoplasm of hepatocytes, epithelial cells, macrophages and fibroblasts. The pattern paralleled that of the endocytic and acidic spaces; there was no clear indication of discrete acidosomes in these mammalian cells. We conclude that the V-H(+)-ATPase in Dictyostelium is distributed among diverse endomembrane organelles and is immunologically cross-reactive with the proton pumps on endocytic vacuoles in mammalian cells.
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PMID:An immunocytochemical analysis of the vacuolar proton pump in Dictyostelium discoideum. 840 7

Amoebae of the eukaryotic microorganism Dictyostelium discoideum were found to contain an interconnected array of tubules and cisternae whose membranes were studded with 15-nm-diameter "pegs." Comparison of the ultrastructure and freeze-fracture behavior of these pegs with similar structures found in other cells and tissues indicated that they were the head domains of vacuolar-type proton pumps. Supporting this identification, the pegs were observed to decorate and clump when broken amoebae were exposed to an antiserum against the B subunit of mammalian vacuolar H(+)-ATPase. The appearance of the peg-rich cisternae in quick-frozen amoebae depended on their osmotic environment: under hyperosmotic conditions, the cisternae were flat with many narrow tubular extensions, while under hypo-osmotic conditions the cisternae ranged from bulbous to spherical. In all cases, however, their contents deep etched like pure water. These properties indicated that the interconnected tubules and cisternae comprise the contractile vacuole system of Dictyostelium. Earlier studies had demonstrated that contractile vacuole membranes in Dictyostelium are extremely rich in calmodulin (Zhu, Q., and M. Clarke, 1992, J. Cell Biol. 118: 347-358). Light microscopic immunofluorescence confirmed that antibodies against the vacuolar proton pump colocalized with anti-calmodulin antibodies on these organelles. Time-lapse video recording of living amoebae imaged by interference-reflection microscopy, or by fluorescence microscopy after staining contractile vacuole membranes with potential-sensitive styryl dyes, revealed the extent and dynamic interrelationship of the cisternal and tubular elements in Dictyostelium's contractile vacuole system. The high density of proton pumps throughout its membranes suggests that the generation of a proton gradient is likely to be an important factor in the mechanism of fluid accumulation by contractile vacuoles.
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PMID:Proton pumps populate the contractile vacuoles of Dictyostelium amoebae. 850 52

Anti-synaptobrevin 2 immunoprecipitates obtained from freshly prepared Triton X-100 extracts of rat synaptosomes contained, in addition to synaptophysin, a 10-kDa band, which we identified by peptide sequencing and Western blotting as the c subunit of the vacuolar proton pump (V-ATPase) also called ductin or mediatophore. Ac39 and Ac116, two other transmembrane subunits of the V0 sector of the V-ATPase, were also found by Western blotting to be enriched in the immunoprecipitates. None of these V-ATPase subunits, or synaptophysin, was present in anti-synaptobrevin 2 immunoprecipitates obtained from frozen-thawed Triton X-100 extracts, which were greatly enriched, instead, in SNAP-25 and syntaxin 1. Accordingly, V-ATPase subunit c was found in anti-synaptophysin immunoprecipitates. Thus, the two complexes appear to be mutually exclusive. Subcellular fractionation of rat brain demonstrated that V-ATPase subunit c is localized with synaptobrevin 2 and synaptophysin in synaptic vesicles. The coprecipitation of V-ATPase subunit c with the synaptobrevin-synaptophysin complex suggests that this interaction may play a role in recruiting the proton pump into synaptic vesicles. Freeze-thawing, which involves a mild denaturing step, may produce a conformational change which dissociates the complex and mimics a change which occurs in vivo as a prerequisite to SNARE complex formation.
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PMID:The V0 sector of the V-ATPase, synaptobrevin, and synaptophysin are associated on synaptic vesicles in a Triton X-100-resistant, freeze-thawing sensitive, complex. 856 78

The clathrin-coated vesicle H+-ATPase is composed of a peripheral catalytic sector (VC) and an integral membrane proton channel (VB), both of which are multiple subunit complexes. This study was conducted to determine if subunit F, previously identified in vacuolar proton pumps of tobacco hornworm and yeast, was present in mammalian pumps. Using a polymerase chain reaction-based strategy, we have isolated and sequenced cDNA clones from bovine and rat brain cDNA libraries. A full-length clone from rat brain encodes a 119-amino acid polypeptide with a predicted molecular mass of 13, 370 Da and with approximately 72 and 49% identity to subunit F of tobacco hornworm and yeast, respectively. Southern and Northern blot analyses indicate that the protein is encoded by a single gene. An anti-peptide antibody, directed against deduced protein sequence, was affinity-purified and shown to react with a 14-kDa polypeptide that is present in a highly purified pump prepared from clathrin-coated vesicles and also isolated VC. When stripped clathrin-coated vacuolars and purified chromaffin granule membranes were treated with KI in the presence of ATP, the 14-kDa subunit was released from both membranes, further indicating that it is part of the peripheral catalytic sector. In addition, direct sequencing of this 14-kDa component of the coated vacuolar proton pump confirmed its identity as a subunit F homologue.
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PMID:Identification of a 14-kDa subunit associated with the catalytic sector of clathrin-coated vesicle H+-ATPase. 862 38


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