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)

We characterized Mg(2+)-dependent ATPase activity in membranes from the renal cortex, the outer and inner stripes of the outer medulla, and papillary vesicles. In all regions, there was Mg(2+)-dependent ATPase activity that was resistant to oligomycin and vanadate and sensitive to N,N'-dicyclohexylcarbodiimide (DCCD), N-ethylmaleimide, and filipin. DCCD-Sensitive Mg(2+)-ATPase activity was highest in the inner stripe of the outer medulla and lowest in the cortex, with intermediate values in the outer stripe of the outer medulla and papilla. The Km for ATP, however, was similar among the different regions of the kidney. DCCD-Sensitive Mg(2+)-ATPase activity was critically dependent upon chloride with Km for Cl- in the range of 2-5 mM. In the presence of ATP, this ATPase was capable of H+ translocation, as assessed by acridine orange quenching. Inhibitors of ATPase activity prevented H+ translocation, which suggests that the Mg(2+)-ATPase represents, at least in part, an H(+)-ATPase. H+ transport was likewise critically dependent upon chloride, with similar Km. The effect of chloride on H+ translocation was blocked by the chloride channel inhibitor, diphenylamine-2 carboxylic acid. In the absence of chloride, H+ transport was abolished, but it could be partially restored by the creation of a favorable electric gradient by K+ and valinomycin. These studies demonstrate that the renal H(+)-ATPase exhibits different activities in various regions of the kidney. The ATPase activity and H+ translocation are critically dependent upon the presence of chloride, which suggests that chloride influences H+ translocation by dissipating the H+ gradient and acting at the catalytic site of the ATPase.
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PMID:ATP-dependent renal H+ translocation: regional localization, kinetic characteristics, and chloride dependence. 138 29

Dysidenin, a hexachlorinated tripeptide-like molecule extracted from the sponge Dysidea herbacea, has lethal effects on fishes and some marine organisms. In an in vitro screening study, this molecule appeared to be a strong inhibitor of iodide transport in dog thyroid slices. Ouabain blocks iodide transport by inhibiting the Na+/K+ ATPase, which sustains the Na+ gradient needed to drive iodide transport. Dysidenin and ouabain block iodide transport with the same kinetics but not by the same mechanisms; dysidenin, unlike ouabain, did not inhibit 86Rb+ uptake or increase its efflux. Inhibitors of chloride channels or carriers did not reduce the T/M value of 131I-, with the exception of phloretin, a relatively nonspecific anion transport blocker. Monesin (or Na+ ionophores) but not dysidenin clearly increased 22Na+ efflux in tracerpreloaded thyroid slices treated with ouabain. This suggests that dysidenin does not act as a chloride channel inhibitor or a Na+ ionophore. Increasing the iodide concentration in the medium decreased the inhibition by dysidenin, suggesting a pseudocompetitive type of effect. To study the structure-activity relationship of dysidenin, several hydrolytic products and synthetic derivatives have been prepared. The data obtained showed that the inhibition is sensitive to stereochemical effects and that the trichloromethyl terminus of the molecule is recognized by the binding site. The presence of only one trichloromethyl terminus is sufficient to exert the inhibitory effect.
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PMID:Inhibition of iodide transport in thyroid cells by dysidenin, a marine toxin, and some of its analogs. 215 65

Advances have been made in the characterization of 5-HT-storing organelles of neurectodermal cells. The parafollicular cell of the thyroid has been used as a model. This cell stores 5-HT, shares many properties with neurons, and can be induced to change its phenotype from endocrine to neuronal by exposure in vitro to NGF. The membranes of isolated parafollicular 5-HT storage vesicles appear to contain a chloride channel that is gated in response to stimulation of the cells by secretogogues. Opening of this channel permits the interior of the vesicle to acidify in response to the action of a H+ ATPase in the vesicular membrane. Development of a delta psi appears to limit acidification of the vesicular interior when the chloride conductance is low. Transmembrane transport of 3H-5-HT into parafollicular vesicle is inhibited by dissipating the delta pH across the granular membranes. The physiological significance of the ability of parafollicular vesicles to modify the internal pH of their 5-HT-storing organelles remains to be determined. Like the synaptic vesicles of central and peripheral serotonergic neurons parafollicular vesicles contain a specific 5-HT binding protein, SBP. 5-HT storage organelles and SBP have been found in medullary thyroid carcinoma (MTC) cells, a tumor line derived from parafollicular cells. The cell biology of SBP is now under study utilizing the MTC cells.
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PMID:Serotonin-storing secretory vesicles. 225 32

With immunocytochemistry, we have determined distribution of sodium, potassium-adenosine triphosphatase (Na+, K+-ATPase) and of three isoenzymes of carbonic anhydrase (CA) and have shown absence of the chloride channel, Band 3 protein, in the genital tract of female rodents. Staining for Na+,K+-ATPase was strongest in the ampullary oviduct and uterine glands in the mouse. In the mouse and rat ovary, immunostaining evidenced CA I, II, and III in theca interna cells where the enzyme could affect the pH of follicular fluid. The zona pellucida of the ovary and cytoplasmic foci in follicular granulosa cells stained for content of only CA I in mouse ovary, suggesting synthesis of a zona pellucida component by granulosa cells. CA II in mouse oviductal epithelium increased from the negative infundibulum to the variably positive ampulla and isthmus to the uniformly positive interstitial segment. The content of CA III varied inversely to that of CA II. The prevalence of CA II-positive cells apparently corresponded with that of nonciliated cells, whereas abundance of CA III-positive cells concurred with that of ciliated cells in regions of the mouse oviduct. The rat oviduct lacked CA II but, like that of the mouse, showed CA III in the proximal region. The staining for CA II in surface epithelium exceeded the reactivity in glandular epithelium in the mouse uterus, except during estrus. In contrast, rat uterus evidenced CA II in glandular but not surface epithelium. These results testify to possible significance of various ion transport mechanisms for biologic activities of diverse cells in the female genital tract.
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PMID:Immunocytochemistry of ion transport mediators in the genital tract of female rodents. 245 38

1. The effects of prostaglandin E2 (PGE2) on ion transport were investigated in guinea-pig isolated gastric mucosa. 2. Under resting conditions the mucosa produced a short-circuit current (SCC), the majority of which could be attributed to electrogenic chloride secretion. This interpretation was confirmed by the dependence of the basal SCC on extracellular chloride, and inhibition by the chloride channel blocker, diphenylamine-2-carboxylate. The mucosa also exhibited low rates of acid secretion and of sodium and rubidium absorption. 3. PGE2 stimulated an increase in net chloride secretion which was more than sufficient to account for the concomitant increase in SCC. As with the basal SCC, the SCC response to PGE2 was chloride dependent and inhibited by diphenylamine-2-carboxylate. PGE2 also significantly increased acid secretion and net rubidium absorption, but these changes were not sufficient to account for SCC. 4. The H+-K+-ATPase inhibitor, omeprazole, inhibited basal and PGE2-stimulated acid secretion, but did not modify the effects the PGE2 on net chloride secretion, SCC or conductance, suggesting that these effects of PGE2 were not related to changes in gastric acid secretion. 5. Both basal and PGE2-stimulated SCC were dependent on extracellular sodium and inhibited by ouabain, indicating the importance of a sodium gradient and the Na+-K+-ATPase in maintaining the electrogenic properties of the mucosa. 6. These results are consistent with the view that PGE2 stimulates electrogenic chloride secretion in guinea-pig gastric mucosa, and provide an ionic basis for the stimulation of secretion of sodium and chloride by prostaglandins observed in mammalian gastric mucosa in vivo.
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PMID:Stimulation of electrogenic chloride secretion by prostaglandin E2 in guinea-pig isolated gastric mucosa. 245 57

Secretory granules of sheep thyroid parafollicular cells contain serotonin, a serotonin-binding protein, and calcitonin. Parafollicular cells, isolated by affinity chromatography, were found to secrete serotonin when activated by thyrotropin (TSH) or elevated [Ca2+]e. TSH also induced a rise in [Ca2+]i. We studied the effect of these secretogogues on the pH difference (delta pH) across the membranes of the secretory granules of isolated parafollicular cells. The trapping of the weak bases, acridine orange or 3-(2,4 dinitro anilino)-3'-amino-N-methyl dipropylamine (DAMP), within the granules was used to evaluate delta pH. In contrast to lysosomes, which served as an internal control, the secretory granules of resting parafollicular cells displayed a limited and variable ability to trap either acridine orange or 3-(2,4 dinitro anilino)-3'-amino-N-methyl dipropylamine; however, when parafollicular cells were stimulated with TSH or elevated [Ca2+]e, the granules acidified. Weak base trapping was also used to evaluate the ATP-driven H+ translocation into isolated parafollicular granules. The isolated parafollicular granules did not acidify in response to addition of ATP unless their transmembrane potential was collapsed by the K+ ionophore, valinomycin. Secretory granules isolated from TSH-treated parafollicular cells had a high chloride conductance than did granules isolated similarly from untreated cells. Furthermore, ATP-driven H+ translocation into parafollicular granules isolated from TSH-stimulated parafollicular cells occurred even in the absence of valinomycin. These results demonstrate that secretogogues can regulate the internal pH of the serotonin-storing secretory granules of parafollicular cells by opening a chloride channel associated with the granule membrane. This is the first demonstration that the pH of secretory vesicles may be modified by altering the conductance of a counterion for the H+ translocating ATPase.
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PMID:Thyrotropin induces the acidification of the secretory granules of parafollicular cells by increasing the chloride conductance of the granular membrane. 246 47

Rat submandibular cells treated with methylcholanthrene are able to be propagated in continuous culture while retaining beta-adrenergic responsiveness. A specific clone, RSMT-A5, has been isolated and studied in detail. RSMT-A5 cells possess beta-adrenergic receptors (BARS) as judged by [3H]-dihydroalprenolol ([3H]-DHA) binding studies. [3H]-DHA binds to RSMT-A5 membranes in a specific and saturable manner with respect to time and [3H]-DHA concentration. Specific binding is saturable within three min of incubation, and a Scatchard analysis reveals a single class of high affinity binding sites with an equilibrium dissociation constant of 0.62 +/- 0.03 nM and a receptor density of 101 +/- 4 fmole/mg protein. Antagonist competition studies indicate that the BARs are primarily of the beta 2-subtype. The BARs are functional since isoproterenol stimulation results in an increased intracellular cAMP content, marked morphological change, and decreased cell volume and chloride content. These same responses can be evoked by treating RSMT-A5 cells with 8-bromo-cAMP. Ion transport inhibitors such as bumetanide (an inhibitor of Na/K/Cl cotransport), SITS and DIDS (inhibitors of chloride-bicarbonate exchange), amiloride (an inhibitor of Na-H exchange), ouabain (an inhibitor of Na/K-ATPase), and dipyridamole and 9-anthracene carboxylic acid (chloride channel blockers) fail to inhibit the isoproterenol-stimulated change in chloride content. The effects of either isoproterenol or 8-bromo-cAMP on both chloride content and cell volume can be inhibited by the chloride channel blocker N-phenylanthranilic acid, however. Taken together, our results indicate that RSMT-A5 cells possess a beta-adrenergic receptor system which controls intracellular volume and chloride content by modulating transport processes that are 1) cAMP-responsive and 2) inhibitable by the putative chloride channel blocker N-phenylanthranilic acid.
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PMID:Beta-adrenergic control of cell volume and chloride transport in an established rat submandibular cell line. 256 99

Polyclonal rabbit antibodies were raised against 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS), an inhibitor of a variety of anion transport proteins. These antibodies specifically recognize SITS-reacted erythrocyte band 3 in immunoprecipitations and Western blots. In Western blots of SITS-reacted membrane proteins derived from vesicles of the electric organ of Torpedo californica (known to express a SITS-sensitive Cl- channel) the antibodies recognized two major species of approximately 93 kDa and approximately 105 kDa. The approximately 93 kDa protein was identified as the alpha-subunit of the Na,K-ATPase. The approximately 105 kDa protein (designated sp105) is a glycoprotein which binds to wheat-germ agglutinin and concanavalin A and is present as a disulphide-linked homodimer under non-reducing conditions. A partial amino acid sequence and a polyclonal antibody were used to clone the corresponding cDNA. sp105 is encoded in electroplax by two abundant mRNAs of approximately 6 and approximately 6.8 kb. A hybridizing mRNA of approximately 5 kb was over 200-fold and over 500-fold less abundant in brain and heart respectively. Sequence analysis of the cDNA predicted a novel protein of 697 amino acids containing eight potential N-linked glycosylation sites. Analysis of hydrophobicity indicated the presence of at least one, and possibly three, putative membrane-spanning domains. When expressed from the Sp6 message in Xenopus laevis oocytes, the protein was inserted into membranes, glycosylated and processed to form a dimer. However, no increase in 36Cl uptake or in membrane conductance could be detected. We found no effect of hybrid depleting the specific message on expression of the Torpedo electroplax Cl- channel in oocytes. Thus we conclude that this novel electroplax membrane protein is probably not a functional part of the chloride channel.
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PMID:Primary structure of a novel 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS)-binding membrane protein highly expressed in Torpedo californica electroplax. 277 1

In myotonic ADR mice that are homozygous for a defect in the muscular chloride channel gene adr/Clc-1, the hyperexcitability of fast muscles is associated with secondary changes in gene expression and fibre type composition. cDNA clones derived from a set of genes down regulated in fast muscles of the myotonic ADR mouse were isolated by a subtractive cloning procedure. A total of 1200 clones were analysed for high expression in fast muscle of wild type and low expression in mutant mouse. Differential transcript levels were verified by northern blot hybridizations. The identities of the corresponding transcripts were determined by sequencing as myosin heavy chain IIB, alpha-tropomyosin, troponin C, a Ca2+ ATPase and parvalbumin mRNAs. Of these, mRNAs for parvalbumin and myosin heavy chain IIB were drastically downregulated in myotonic muscle (to < 10% of control). A full length cDNA clone for skeletal muscle alpha-tropomyosin was homologous to the mouse fibroblast tropomyosin isoform 2, except for the portion encoding the alpha-tropomyosin specific amino acids 258-284. A cDNA derived from the 1100 nucleotide parvalbumin transcript was cloned and the sequence for the as yet unknown 3' extended trailer, generated by alternative polyadenylation, was determined.
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PMID:Subtractive cDNA cloning as a tool to analyse secondary effects of a muscle disease. Characterization of affected genes in the myotonic ADR mouse. 752 80

Gastric intracellular tubulovesicles fuse with the apical membrane upon histamine stimulation. Disulfide cross-linking of isolated gastric tubulovesicles by cupper-o-phenanthroline (CuP) opened the chloride channel in the vesicles. A functional monoclonal antibody raised against H+,K(+)-ATPase of gastric vesicles inhibited both the enzyme activity and the CuP-induced opening of the chloride channel. This fact indicates that the chloride channel is part of the function of the ATPase. Another evidence which supports the above concept that both the pump and the chloride channel coexist in the same molecule was obtained in this study. The conformation of H+,K(+)-ATPase was changed in the direction of E2 form by incubation with SCH 28080 or low concentrations of K+. SCH 28080 is an H+,K(+)-ATPase specific inhibitor and binds to the high affinity K+ site. Both SCH 28080 and K+ inhibited the channel opening, indicating that the channel opening by the S-S cross-linking depends on the conformational state of the enzyme.
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PMID:The apical chloride channel as part of the function of gastric H+,K(+)-ATPase. 775 19


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