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)

Cytoskeleton membrane associations are important for a variety of cellular functions. The anion exchanger of erythrocytes (AE1) and Na+,K(+)-ATPase of polarized epithelial cells provide well studied examples of how integral membrane proteins are anchored via the linker molecule ankyrin to the spectrin-based membrane cytoskeleton. In the present study we have generated several recombinant fragments of the large (third) cytoplasmic domain (CD3) of Na+,K(+)-ATPase to define binding sites of ankyrin on CD3 at a molecular level. We provide evidence that a cluster of four amino acids, ALLK, is essential for binding of ankyrin to both recombinant CD3 and to native Na+,K(+)-ATPase. Once bound, conformational changes might uncover further binding sites for ankyrin on Na+,K(+)-ATPase. A motif related to the ALLK cluster is also present in the cytoplasmic domain of AE1 where this sequence (ALLLK) turned out to be also important for ankyrin binding. These motifs are highly conserved during evolution of both Na+,K(+)-ATPase and AE1, further underlining their potential role in cytoskeleton to membrane linkage.
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PMID:Identification of a binding motif for ankyrin on the alpha-subunit of Na+,K(+)-ATPase. 853 Mar 98

The kidneys of mice (CAR2-null mice) that are genetically devoid of carbonic anhydrase type II (CAII) were screened by immunocytochemistry with antibodies that distinguish intercalated and principal cells. Immunofluorescent localization of the anion exchanger AE1 and of the 56-kDa subunit of the vacuolar H(+)-adenosinetriphosphatase (H(+)-ATPase) was used to identify intercalated cells, while the AQP2 water channel was used as a specific marker for principal cells of the collecting duct. The CAII deficiency of the CAR2-null mice was first confirmed by the absence of immunofluorescent staining of kidney sections exposed to an anti-CAII antibody. Cells positive for AE1 and H(+)-ATPase were common in all collecting duct regions in normal mice but were virtually absent from the inner stripe of the outer medulla and the inner medulla of CAR2-null mice. The number of positive cells was also reduced threefold in the cortical collecting duct of CAR2-null animals compared with normal mice. In parallel, the percentage of AQP2-positive cells was correspondingly increased in the collecting tubules of CAII-deficient mice, whereas the total number of cells per tubule remained unchanged. These results suggest that intercalated cells are severely depleted and are replaced by principal cells in CAII-deficient mice. Quantitative analysis and double staining showed that, in the cortex, both type A and type B intercalated cells are equally affected. Elucidation of the mechanism(s) responsible for this phenotype will be of importance in understanding the origin and development of intercalated cells in the kidney.
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PMID:Depletion of intercalated cells from collecting ducts of carbonic anhydrase II-deficient (CAR2 null) mice. 859 70

Osteoclasts attach to the bone surface and resorb bone by secreting protons into an isolated subosteoclastic compartment. Previous studies have shown the presence of a vacuolar type H+-ATPase, and a functional Cl--HCO3- anion exchanger in the osteoclast. In the present studies, using a monoclonal antibody to the 31-kDa subunit of H+-ATPase and a rabbit antiserum to the erythrocyte band-3 protein (Cl--HCO3- anion exchanger) we have immunocytochemically localized the respective pumps in bone sections obtained from chickens fed a normal or a calcium-deficient diet for 4 weeks. Our results indicate that although H+-ATPase is either evenly distributed throughout the osteoclast or is more polarized at its ruffled membrane juxtaposed to the bone surface, the band-3 protein immunoreactivity is always localized to the plasma membrane which is not attached to the bone surface (basolateral membrane). Four weeks of a calcium-deficient diet resulted in a significant increase in the percentage of osteoclasts that were polarized for the H+-ATPase pump at their ruffled membrane, and a trend toward increased total number of osteoclasts, although the latter did not reach statistical significance (P = 0.09). These changes were not accompanied by a significant increase in the intensity of staining for H+-ATPase. Band-3 protein immunoreactivity was always prominent, limited to the basolateral membrane, and did not alter with calcium-deficient diet or with changes in the degree of H+-ATPase polarization.
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PMID:Immunocytochemical localization of vacuolar H+-ATPase and Cl--HCO3- anion exchanger (erythrocyte band-3 protein) in avian osteoclasts: effect of calcium-deficient diet on polar expression of the H+-ATPase pump. 866 62

This study investigates the mechanism of magnesium (Mg2+) transport (efflux) from the exocrine rat pancreas. Permeabilized pancreatic acini were loaded with Mg2+ by employing a high-Mg2+ (12 mM) buffer containing A23187 (6 microM). Net Mg2+ efflux was measured using the technique of atomic absorbance spectrophotometry. Incubation of preloaded acini in a buffer deficient in Mg2+ resulted in a large and time-dependent release of Mg2+ with maximal efflux occurring within 40 min. Pretreatment of loaded acini with bumetanide, SITS or ouabain had no significant effect on Mg2+ efflux. In contrast, when acini were pretreated with 10 mM dinitrophenol, 10(-4) M amiloride, 1 mM lidocaine or 1 mM quinidine there were significant (P < 0.001) decreases in net Mg2+ efflux. Replacement of extracellular sodium [Na+]o with either N-methyl-D-glucamine (NMDG), Tris or choline resulted in a significant (P < 0.001) inhibition of Mg2+ efflux. The results of this study indicate that Mg2+ transport (efflux) in pancreatic acinar cells may not be associated with the Na(+)-K(+)-ATPase, the Na(+)-K(+)-Cl- cotransporter or the anion exchanger, but with a Na(2+)-sensitive Mg2+ transport system.
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PMID:Characterization of a sodium-dependent magnesium efflux from magnesium-loaded rat pancreatic acinar cells. 873 71

The cortical collecting duct (CCD) mediates net secretion or reabsorption of protons according to systemic acid/base status. Using indirect immunofluorescence, we examined the localization and abundance of the vacuolar H(+)-ATPase and the AE1 anion exchanger in intercalated cells (IC) of rat kidney connecting segment (CNT) and CCD during acute (6 hr) metabolic (NH4Cl) acidosis and respiratory (NaHCO3) alkalosis. AE1 immunostaining intensity quantified by confocal microscopy was elevated in metabolic acidosis and substantially reduced in metabolic alkalosis. AE1 immunostaining was restricted to Type A IC in all conditions, and the fraction of AE1+IC was unchanged in CNT and CCd. Metabolic acidosis was accompanied by redistribution of H(+)-ATPase immunostaining towards the apical surface of IC, and metabolic alkalosis was accompanied by H(+)-ATPase redistribution towards the basal surface of IC. Therefore, acute metabolic acidosis produced changes consistent with increased activity of Type A IC and decreased activity of Type B IC, whereas acute metabolic alkalosis produced changes corresponding to increased activity of Type B IC and decreased activity of Type A IC. These data demonstrate that acute systemic acidosis and alkalosis modulate the cellular distribution of two key transporters involved in proton secretion in the distal nephron.
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PMID:Regulation of AE1 anion exchanger and H(+)-ATPase in rat cortex by acute metabolic acidosis and alkalosis. 899 26

A simple method for antigen retrieval in tissue sections and cell cultures is described. Because many antibodies recognize denatured proteins on western blots, but are poorly reactive by immunocytochemistry, the effect of applying sodium dodecyl sulfate (SDS) to cryostat sections of tissues and to cell cultures prior to immunostaining was examined. In many cases, a 5-min pretreatment with 1% SDS produced a dramatic increase in staining intensity by indirect immunofluorescence. Among the antibodies tested that showed a positive effect of SDS were an anti-Na/K-ATPase monoclonal antibody, an anti-AE1/2 anion exchanger polyclonal antipeptide antibody, a monoclonal anti-caveolin antibody, and an anti-rab4 monoclonal antibody. In other cases, including antibodies against gp330, aquaporin 1, and aquaporin 2, no effect of SDS was detected. The results show that SDS treatment can be used as a simple method of antigen retrieval in cryostat sections and on cultured cells. In some cases, antigens were not detectable without pretreatment with SDS.
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PMID:Antigen retrieval in cryostat tissue sections and cultured cells by treatment with sodium dodecyl sulfate (SDS). 907 83

The effect of NO3- on intracellular pH (pHi) was assessed microfluorimetrically in mammalian cells in culture. In cells of human, hamster, and murine origin addition of extracellular NO3- induced an intracellular acidification. This acidification was eliminated when the cytosolic pH was clamped using ionophores or by perfusing the cytosol with highly buffered solutions using patch-pipettes, ruling out spectroscopic artifacts. The NO3-- induced pH change was not due to modulation of Na+/H+ exchange, since it was also observed in Na+/H+ antiport-deficient mutants. Though NO3- is known to inhibit vacuolar-type (V) H+-ATPases, this effect was not responsible for the acidification since it persisted in the presence of the potent V-ATPase inhibitor bafilomycin A1. NO3-/HCO3- exchange as the underlying mechanism was ruled out because acidification occurred despite nominal removal of HCO3-, despite inhibition of the anion exchanger with disulfonic stilbenes and in HEK 293 cells, which seemingly lack anion exchangers (Lee, B. S., R.B. Gunn, and R.R. Kopito. 1991. J. Biol. Chem. 266:11448- 11454). Accumulation of intracellular NO3-, measured by the Greiss method after reduction to NO2-, indicated that the anion is translocated into the cells along with the movement of acid equivalents. The simplest model to explain these observations is the cotransport of NO3- with H+ (or the equivalent counter-transport of NO3- for OH-). The transporter appears to be bi-directional, operating in the forward as well as reverse directions. A rough estimate of the fluxes of NO3- and acid equivalents suggests a one-to-one stoichiometry. Accordingly, the rate of transport was unaffected by sizable changes in transmembrane potential. The cytosolic acidification was a saturable function of the extracellular concentration of NO3- and was accentuated by acidification of the extracellular space. The putative NO3--H+ cotransport was inhibited markedly by ethacrynic acid and by alpha-cyano-4-hydroxycinnamate, but only marginally by 4, 4'-diisothiocyanostilbene-2,2' disulfonate or by p-chloromercuribenzene sulfonate. The transporter responsible for NO3--induced pH changes in mammalian cells may be related, though not identical, to the NO3--H+ cotransporter described in Arabidopsis and Aspergillus. The mammalian cotransporter may be important in eliminating the products of NO metabolism, particularly in cells that generate vast amounts of this messenger. By cotransporting NO3- with H+ the cells would additionally eliminate acid equivalents from activated cells that are metabolizing actively, without added energetic investment and with minimal disruption of the transmembrane potential, inasmuch as the cotransporter is likely electroneutral.
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PMID:NO3--induced pH changes in mammalian cells. Evidence for an NO3--H+ cotransporter. 923 11

H,K-ATPase from gastric mucosa is responsible for HCI secretion in the gastric lumen and is a member of the P-type ATPase family. The structure of enzyme subunits, their functions and topology, the mechanism of ATP hydrolysis and transport function of the enzyme, its specific inhibitors, and the success of their pharmacological application are reviewed. The methods for isolation of membrane fractions with H,K-ATPase activity and attempts for solubilization and purification of the enzyme are described. Data demonstrating the presence of H,K-ATPase in other tissues are considered. Information about other enzyme systems of parietal cells involved in transepithelial transport of HCl (the Cl- and K-channels of the apical membrane, the HCO3-/Cl- anion exchanger and Na+/H+ cation exchanger of the basolateral membrane) is presented. Mechanisms of activation of acid secretion by parietal cells via gastrin, acetylcholine, and histamine receptors and the role of cytoskeletal proteins in activation are reviewed.
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PMID:H,K-ATPase and acid secretion control in gastric mucosa. 946 28

Cold preservation of kidneys is commonly used in human transplantation and in vitro studies. However, although disruption of the cytoskeleton by cold has been demonstrated in cultured cells, the effect of cold treatment on intact kidney is poorly understood. In this study, specific antibodies were used to examine the effect of hypothermia on the cytoskeletal network and the trafficking of some membrane proteins in the urinary tubule. Rat kidneys were cut into thin slices (approximately 0.5 mm) that were divided into several groups: (1) some were immediately fixed in paraformaldehyde, sodium periodate, and lysine (PLP); (2) some were stored at 4 degrees C for 15 min or 4 h before being fixed in cold PLP; or (3) after 4 h cold treatment, some slices were rewarmed to 37 degrees C for 15, 30, and 60 min in a physiologic solution, pH 7.4, and were then fixed in warm PLP. Immunofluorescence staining revealed an almost complete disruption of the microtubule network in proximal tubules after 15 min cold treatment, whereas microtubules in other segments were affected after 4 h. A partial recovery of the microtubule network was observed after 60 min rewarming. In contrast, actin filaments seemed to be resistant to cold treatment. gp330, aquaporin-2, H+ ATPase, and the AE1 anion exchanger were all relocated into numerous vesicles that were distributed throughout the cytoplasm after hypothermia followed by rewarming, whereas Na-K-ATPase retained its basolateral localization. The vasopressin-stimulated insertion of aquaporin-2 water channels into the apical membrane was inhibited during the initial rewarming period after cold exposure. Thus, cold preservation of tissues might impair, at least transiently, the polarized membrane expression and function of some transport proteins in renal epithelial cells.
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PMID:Cold-induced microtubule disruption and relocalization of membrane proteins in kidney epithelial cells. 952 91

When an intercalated epithelial cell line was seeded at low density and allowed to reach confluence, it located the anion exchanger band 3 in the apical membrane and an H+-ATPase in the basolateral membrane. The same clonal cells seeded at high density targeted these proteins to the reverse location. Furthermore, high density cells had vigorous apical endocytosis, and low density cells had none. The extracellular matrix of high density cells was capable of inducing apical endocytosis and relocation of band 3 to the basolateral membrane in low density cells. A 230-kDa extracellular matrix (ECM) protein termed hensin, when purified to near-homogeneity, was able to reverse the phenotype of the low density cells. Antibodies to hensin prevented this effect, indicating that hensin is necessary for conversion of polarity. We show here that hensin was synthesized by both low density and high density cells. Whereas both phenotypes secreted soluble hensin into their media, only high density cells localized it in their ECM. Analysis of soluble hensin by sucrose density gradients showed that low density cells secreted monomeric hensin, and high density cells secreted higher order multimers. When 35S-labeled monomeric hensin was added to high density cells, they induced its aggregation suggesting that the multimerization was catalyzed by surface events in the high density cells. Soluble monomeric or multimeric hensin did not induce apical endocytosis in low density cells, whereas the more polymerized hensin isolated from insoluble ECM readily induced it. These multimers could be disaggregated by sulfhydryl reagents and by dimethylmaleic anhydride, and treatment of high density ECM by these reagents prevented the induction of endocytosis. These results demonstrate that hensin, like several ECM proteins, needs to be precipitated in the ECM to be functional.
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PMID:Only multimeric hensin located in the extracellular matrix can induce apical endocytosis and reverse the polarity of intercalated cells. 1036 6


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