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 gastric H+/K(+)-transporting adenosine triphosphatase (H+/K+ ATPase) (proton pump) consists of a catalytic alpha-subunit and a recently proposed 60-90-kDa glycoprotein beta-subunit. Using dog gastric membranes as the antigen, we have produced two murine monoclonal antibodies, 4F11 (IgG1) and 3A6 (IgA), which are specific for the 60-90-kDa glycoprotein. The monoclonal antibodies (1) specifically stained the cytoplasm of unfixed and formalin-fixed dog gastric parietal cells; (2) specifically reacted by ELISA with gastric tubulovesicular membranes; (3) recognised epitopes located on the luminal face of parietal cell tubulovesicular membranes, the site of the proton pump, by immunogold electron microscopy; (4) immunoblotted a 60-90-kDa molecule from tubulovesicular membranes and a 35-kDa component from peptide N-glycosidase-F-treated membrane extracts; (5) immunoblotted the 60-90-kDa parietal cell autoantigen associated with autoimmune gastritis and pernicious anemia, purified by chromatography on parietal cell autoantibody- or tomato-lectin-Sepharose 4B affinity columns, and the 35-kDa protein core of this autoantigen; this autoantigen has amino acid sequence similarity to the beta-subunit of the related Na+/K(+)-transporting adenosine triphosphatase (Na+/K+ ATPase) [Toh et al. (1990) Proc. Natl Acad. Sci. 87, 6418-6422]; (6) co-precipitated a molecule of 95 kDa with the 60-90-kDa molecule from 125I-labelled detergent extracts of dog tubulovesicular membranes; and (7) co-purified the catalytic alpha-subunit of the H+/K+ ATPase with the 60-90-kDa molecule by immunoaffinity chromatography of tubulovesicular membrane extracts on a monoclonal antibody 3A6-Sepharose 4B column, indicating a physical association between the two molecules. These results provide further evidence that the 60-90-kDa glycoprotein is the beta-subunit of the gastric H+/K+ ATPase. We conclude that the monoclonal antibodies specifically recognise luminal epitopes on the 35-kDa core protein of the 60-90-kDa beta-subunit of the gastric proton pump, a major target molecule in autoimmune gastritis and pernicious anaemia. These monoclonal antibodies will be valuable probes to study the structure and function of this associated beta-subunit, as well as the ontogeny of the gastric proton pump.
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PMID:Monoclonal antibodies specific for the core protein of the beta-subunit of the gastric proton pump (H+/K+ ATPase). An autoantigen targetted in pernicious anaemia. 170 13

Ion transport cells in gerbil inner ear were differentiated histochemically by staining glycoconjugates (GCs) with a battery of horseradish peroxidase-conjugated lectins. Strong staining with PSA and LCA showed a high content of N-linked oligosaccharides in transport cell GCs. Reactivity with PHA-L and PHA-E identified GC with triantennary and with bisected biantennary N-linked oligosaccharides, respectively, in these cells. High affinity for DSA and PWM demonstrated abundant N-acetyl lactosamine in N-linked side chains. Ion transporting epithelial cells reacting with lectins specific for N-linked oligosaccharides included strial marginal cells and outer sulcus cells of the cochlea and dark cells, transitional cells, and planum semilunatum cells of the vestibular system. In general, all of the inner ear transport epithelial cells revealed a similar lectin binding profile, with the one exception that SBA reacted strongly with ion transporting cells in the vestibular system but only weakly with those in the cochlea. Fibrocytes specialized for ion transport located in distinct areas in the suprastrial and inferior regions of the spiral ligament also stained with lectins that demonstrate N-glycosylation. However, transport fibrocytes differed from transport epithelial cells in two ways. First, they reacted e with HPA, DBA, VVA, and SJA specific for O-linkages and second, they failed to react with UEA I. The staining pattern for N-glycosylated GC resembled that for Na+, K(+)-ATPase in inner ear, suggesting a relationship between these constituents.
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PMID:Distribution of glycoconjugates in ion transport cells of gerbil inner ear. 184 71

Two major types of intercalated cells (IC) have been previously defined in rabbit collecting duct: alpha-cells have a basolateral band 3-like anion exchanger and secrete H+, whereas beta-cells bind peanut agglutinin (PNA) apically and are believed to secrete HCO3-. To further define IC types, we double-labeled kidney sections with anti-H(+) -ATPase antibodies and with either an anti-band 3 antibody or PNA. We found four patterns of staining: 1) IC with apical H(+)-ATPase and basal band 3, a configuration consistent with ongoing H+ secretion, which prevailed in the inner stripe of outer medulla (OMCDi); 2) diffuse H(+)-ATPase labeling across the cell and basal band 3, which was most numerous in the outer stripe of outer medulla (OMCDo); 3) IC with "bright" apical peanut lectin, diffuse H(+)-ATPase, and no band 3, which was abundant in the cortical collecting duct (CCD) and probably represents HCO3(-)-secreting cells; and 4) "hybrid" cells with various staining combinations (e.g., apical lectin binding and apical H(+)-ATPase), which although they are uncommon, were seen in the CCD. Consistent with this immunocytochemical finding of hybrid cells, cell-sorting studies on isolated CCD IC showed that 6-18% of PNA-positive cells also stained positively for band 3. We conclude that 1) band 3-positive IC in the OMCD vary axially. Most OMCDi IC are probably active proton secretors, whereas up to one-half of OMCDo IC may be latent H+ secretors. 2) The diffuse H(+)-ATPase pattern in putative beta-cells differs from comparable results in the rat and is not consistent with a "reversed" alpha-cell. HCO3- secretion by beta-cells may be driven by an H+ extrusion mechanism other than the alpha-cell pump re-sorted to the basolateral membrane. 3) The possibility of hybrid cells that might combine alpha- and beta-cell transport proteins suggests a mechanism for functional reversal of collecting duct IC polarity.
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PMID:Colocalization of H(+)-ATPase and band 3 anion exchanger in rabbit collecting duct intercalated cells. 184 62

Plasma membrane receptors are crucial for nonself tissue recognition. Using concanavalin A (Con A), wheat germ agglutinin, peanut agglutinin, soybean agglutinin (SBA), and winged pea agglutinin, five lectin-binding receptor molecules have been recognized on the plasma membrane of the granulocyte (immunocyte) of the horseshoe crab, Limulus polyphemus. Only Con A and SBA caused capping of surface receptors. On the basis of the known functions of these lectin-binding receptor molecules in other invertebrates and vertebrates, their roles in phagocytosis, encapsulation, signaling, and possibly in complement pathway activation are postulated. In addition to lectin-binding receptors, Na+,K(+)-ATPase and acetylcholinesterase were detected on the plasma membrane. Because Limulus dates back to some 200 million years, the antiquity of these molecules is suggested. Furthermore, some of the lectin-binding surface receptors have the potential to be used as markers to separate different kinds of hemocytes in higher arthropods and to distinguish between normal and neoplastic cells in humans.
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PMID:Lectin-binding receptors, Na+,K(+)-ATPase, and acetylcholinesterase on immunocyte plasma membrane of Limulus polyphemus. 184 29

In order to refine further our structural model of the coated vesicle (H+)-ATPase (Arai, H., Terres, G., Pink, S., and Forgac, M. (1988) J. Biol. Chem. 263, 8796-8802), we have extended our structural analysis to identify peripheral and glycosylated subunits of the pump as well as to identify subunits which are in close proximity in the native (H+)-ATPase complex. Treatment of the purified, reconstituted (H+)-ATPase with 0.30 M KI in the presence or absence of ATP or MgATP results in the release of the 73-, 58-, 40-, 34-, and 33-kDa subunits, leaving behind the 100-, 38-, 19-, and 17-kDa subunits in the membrane. Because the former group of polypeptides is released from the membrane in the absence of detergent, they correspond to peripheral membrane proteins. To determine which subunits are in close proximity, cross-linking of the purified (H+)-ATPase was carried out using the cleavable, bifunctional amino reagent 3,3'-dithiobis(sulfosuccinimidylpropionate) followed by two-dimensional gel electrophoresis. These studies indicate that contact regions exist between the 73- and 58-kDa subunits as well as between the 17-kDa subunit and the 40-, 34-, and 33-kDa subunits. To test for glycosylation of the (H+)-ATPase, the detergent-solubilized complex was treated with neuraminidase followed by electrophoresis and blotting using a peanut lectin/horseradish peroxidase conjugate. Galactose-inhibitable staining of the 100-kDa subunit, together with affinity chromatography of the intact (H+)-ATPase on peanut lectin agarose, indicates that the 100-kDa subunit is glycosylated, most likely at a site exposed on the luminal side of the membrane. These results, together with those presented in the preceding paper (Adachi, I., Arai, H., Pimental, R., and Forgac, M. (1990) J. Biol. Chem. 265, 960-966), were used in the construction of a refined model of the coated vesicle (H+)-ATPase.
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PMID:Dissociation, cross-linking, and glycosylation of the coated vesicle proton pump. 196 52

Autoantibodies in the sera of patients with pernicious anemia recognize, in addition to the alpha subunit of the gastric H+/(+)-ATPase, an abundant gastric microsomal glycoprotein of apparent Mr 60,000-90,000. Herein we have colocalized the glycoprotein and the alpha subunit of the gastric H+/K(+)-ATPase to the tubulovesicular membranes of the parietal cell by immunogold electron microscopy. Moreover, the glycoprotein and the alpha subunit were coimmunoprecipitated, and copurified by immunoaffinity chromatography, with an anti-glycoprotein monoclonal antibody. The pig glycoprotein was purified by chromatography on tomato lectin-Sepharose, and five tryptic peptides from the purified glycoprotein were partially sequenced. The complete amino acid sequence, deduced from the nucleotide sequence of overlapping cDNA clones, showed 33% similarity to the sequence of the beta subunit of the pig kidney Na+/K(+)-ATPase. We therefore propose that the 60- to 90-kDa glycoprotein autoantigen is the beta subunit of the gastric H+/K(+)-ATPase and that the alpha and beta subunits of the proton pump are major targets for autoimmunization in autoimmune gastritis.
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PMID:The 60- to 90-kDa parietal cell autoantigen associated with autoimmune gastritis is a beta subunit of the gastric H+/K(+)-ATPase (proton pump). 197 21

The 53-kDa glycoprotein and sarcalumenin (160-kDa glycoprotein) were extracted from rabbit skeletal muscle sarcoplasmic reticulum with EGTA and purified by fractionation on DEAE-Sephadex A-25 and lentil lectin-Sepharose 4B. Sarcalumenin was shown to bind up to 400 nmol of Ca2+/mg of protein at pH 7.5, which is equivalent to binding of approximately 35 mol of Ca2+/mol of protein. The apparent dissociation constant was 300 microM in the presence of 20 mM KCl and 600 microM in 150 mM KCl. The 53-kDa glycoprotein did not bind any Ca2+ under the conditions examined. Immunoblot analysis of isolated sarcoplasmic reticulum subfractions demonstrated the presence of the two glycoproteins in both the longitudinal sarcoplasmic reticulum and the terminal cisternae. Their concentrations were higher, however, in the longitudinal sarcoplasmic reticulum vesicles. Comparative immunoelectron microscopic studies using monoclonal antibodies revealed a codistribution of the 53-kDa glycoprotein with the Ca2(+)-ATPase in all regions of the free sarcoplasmic reticulum. A similar distribution was found for sarcalumenin, although immunolabeling was much weaker. The colocalization of the 53-kDa glycoprotein and sarcalumenin with the Ca2(+)-ATPase and the Ca2+ binding properties of sarcalumenin suggest that the glycoproteins may be involved in the sequestration of Ca2+ in the nonjunctional regions of the sarcoplasmic reticulum.
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PMID:Purification, calcium binding properties, and ultrastructural localization of the 53,000- and 160,000 (sarcalumenin)-dalton glycoproteins of the sarcoplasmic reticulum. 211 42

Cultured inner medullary collecting duct (IMCD) cells have been shown to secrete protons (H+) by two mechanisms: an N-ethylmaleimide- and dicyclohexyl-carbodiimide-sensitive electrogenic H(+)-ATPase or H+ pump, and an amiloride-sensitive, secondary active Na+H+ exchanger. These cells also express Cl-/HCO3- exchange and carbonic anhydrase activity in common with other renal epithelial cells involved in acid-base transport. Video fluorescence microscopy of individual cells using 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein has demonstrated that adjacent-cultured IMCD cells show substantial functional intercellular heterogeneity. The development of H(+)-pumping activity is associated with high-baseline intracellular pH and peanut agglutinin (PNA) affinity, and loss of mitotic activity and of Na+/H+ exchange. The H(+)-pumping activity may be further enhanced by removal of fetal calf serum for 6-54 h or by selecting cells with high PNA affinity. IMCD cells in their most differentiated state form domes, which consistently showed the highest rates of H(+)-pumping activity, as well as high affinity for peanut lectin. When IMCD were plated at low density, domes developed relatively late (2-4 weeks), at which time cells located in the center of nests of contiguously growing cells were quiescent and showed H(+)-pumping activity but no Na+/H+ exchange. On the other hand, dense plating was associated with early development of domes (end of 1st week), at which time adjacent cells showed a high mitotic activity and Na+/H+ exchange, but no H(+)-pumping activity. We speculate that differentiation of IMCD cells results in the development of cell polarity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Differentiation of proton-pumping activity in cultured renal inner medullary collecting duct cells. 216 49

Evidence is presented for the existence of ectoenzymes in rat renal cortical brush-border membrane vesicles that produce adenosine as a final product using either ATP, ADP or AMP as substrate. The enzymes are insensitive to levamisole, ouabain, oligomycin and N-ethylmaleimide, and have absolute requirement for divalent cations with following order of activation Mg2+ greater than Ca2+ greater than Mn2+ greater than Ba2+ greater than Zn2+. At least two separate enzymes can be distinguished. One is capable of hydrolyzing ATP, other nucleoside triphosphates and ADP, but not AMP. The enzyme is insensitive to concanavalin A. The other enzyme hydrolyzes AMP and is strongly inhibited by this lectin. Mg2(+)-stimulated ATP hydrolysis displays saturation kinetics which is not of the simple Michaelis-Menten type, but is biphasic with a high-affinity (K'm = 0.16 mM) and low-affinity site (K'm = 9.0 mM), respectively. The low-affinity site hydrolyzes ATP, ITP and GTP to a similar extent, whereas CTP and UTP with about 40% lower rate. The high-affinity site splits ATP much better than other nucleoside triphosphates. Hydrolysis of ADP follows simple Michaelis-Menten saturation kinetic with apparent Km = 0.38 +/- 0.06 mM. Inhibition, activation and substrate specificity studies indicate that nucleoside triphosphatase and nucleoside diphosphatase may reside on the same protein. Kinetics of the AMP hydrolysis is hyperbolic with apparent Km = 76 +/- 9 microM. The cascade of ectonucleotidases in the brush-border membrane of the proximal tubule may catalyze the degradation of filtered nucleotides into adenosine and phosphate, the compounds which are thereafter probably reabsorbed by separate transport systems.
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PMID:The stepwise hydrolysis of adenine nucleotides by ectoenzymes of rat renal brush-border membranes. 217

The Ca2+ antagonist binding sites associated with the voltage dependent calcium channel in rabbit myocardium were found to distribute with the sarcolemmal Na+ + K+ ATPase and adenylate cyclase activities during subcellular fractionation on sucrose-density gradients. The equilibrium dissociation constants (KD) for the binding of [3H]nitrendipine and [3H]verapamil were 0.31 +/- 0.04 nM and 4.1 +/- 0.5 nM respectively, and displayed an average density of 0.55 +/- 0.05 pmol/mg and 0.4 +/- 0.03 pmol/mg protein respectively for the most enriched membrane fraction. The Ca2+ antagonist binding sites were solubilized from the membranes with the detergent 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate, and specific binding sites for [3H]PN200-110, [3H]verapamil and [3H]diltiazem were isolated on a wheat-germ lectin column. The binding sites for [3H]PN200-110 were enriched about 2,500 fold as compared with the original homogenate and displayed a density of 28.5 +/- 8 pmole/mg protein in the isolated fraction. Sodium dodecyl sulfate gel electrophoresis of the isolated drug binding proteins indicated enrichment of proteins of Mr 170,000, 140,000, 130,000, 100,000 and 53,000. The isolated receptor contained an intrinsic kinase activity that phosphorylated glycoproteins of Mr 170,000 and 53,000. Exogenously added cAMP-kinase stimulated phosphorylation of the 170,000, 100,000, 53,000 and 28,000 Mr glycoproteins in the receptor fraction. The results of this study indicate that the binding sites for [3H]nitrendipine, [3H]PN200-110, [3H]verapamil and [3H]diltiazem residue on glycoprotein(s) which are of sarcolemmal origin, and co-purify together on wheat germ lectin columns. The polypeptide composition of the Ca2+ antagonist binding sites from cardiac muscle appears to be very similar to that of the dihydropyridine receptor in skeletal muscle.
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PMID:Subcellular distribution and isolation of the Ca2+ antagonist receptor associated with the voltage regulated Ca2+ channel from rabbit heart muscle. 244 72


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