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 freshwater cyanobacterium Synechococcus PCC 6311 is able to adapt to grow after sudden exposure to salt (NaCl) stress. We have investigated the mechanism of Na+ transport in these cells during adaptation to high salinity. Na+ influx under dark aerobic conditions occurred independently of delta pH or delta psi across the cytoplasmic membrane, ATPase activity, and respiratory electron transport. These findings are consistent with the existence of Na+/monovalent anion cotransport or simultaneous Na+/H(+)+anion/OH- exchange. Na+ influx was dependent on Cl-, Br-, NO3-, or NO2-. No Na+ uptake occurred after addition of NaI, NaHCO3, or Na2SO4. Na+ extrusion was absolutely dependent on delta pH and on an ATPase activity and/or on respiratory electron transport. This indicates that Na+ extrusion via Na+/H+ exchange is driven by primary H+ pumps in the cytoplasmic membrane. Cells grown for 4 days in 0.5 M NaCl medium, "salt-grown cells," differ from control cells by a lower vmax of Na+ influx and by lower steady-state ratios of [Na+]in/[Na+]out. These results indicate that cells grown in high-salt medium increase their capacity to extrude Na+. During salt adaptation Na+ extrusion driven by respiratory electron transport increased from about 15 to 50%.
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PMID:NMR studies on Na+ transport in Synechococcus PCC 6311. 131 38

Proteins in human red cell hemolysate were purified to determine which of them increase inhibition of the Na,K-ATPase in the presence of 2 microM free Ca. Samples purified 600,000-fold inhibited the Na,K-ATPase of human red cells in a Ca-dependent manner and stimulated the (Ca+Mg)-ATPase. These samples contained two proteins as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): calmodulin (18,000 Mr), which comprised most (greater than 90%) of the total protein, and an unidentified protein of approximately 13,000 Mr. Both proteins were a distinctive light yellow when stained with silver. Calmodulin from bovine testes also inhibited the Na,K-ATPase and stimulated the (Ca+Mg)-ATPase. This preparation also contained two proteins as analyzed by SDS-PAGE: calmodulin (95 to 99% of the total protein) and another protein of approximately 13,000 Mr (1 to 5% of the total protein). Both were light yellow when stained with silver. Since the amount of red cell protein was limited, the remainder of the study was carried out with the bovine testes preparation. Heating the testes preparation decreased, but did not abolish, inhibition of the Na,K-ATPase and reduced stimulation of the (Ca+Mg)-ATPase. When corrected for denatured calmodulin, both heated and unheated proteins increased inhibition of the Na,K-ATPase to the same extent. The Na,K-ATPase was inhibited at 2 microM free Ca in a dose-dependent manner over a range of 15 to 100 nM calmodulin. To establish if the inhibition was due to the calmodulin or the 13,000 Mr protein, both were electroeluted after SDS-PAGE. Electroeluted calmodulin stimulated the (Ca+Mg)-ATPase and increased Ca inhibition of the Na,K-ATPase. Electroeluted amounts of the smaller Mr protein slightly stimulated the (Ca+Mg)-ATPase, but had no effect on the Na,K-ATPase. This protein was digested with cyanogen bromide, partially sequenced, and thereby identified as a fragment of calmodulin. We conclude that intact calmodulin increases inhibition of the Na,K-ATPase at 2 microM free Ca. We suggest that calmodulin is part of a mechanism mediating the effects of physiological free Ca on the Na,K-ATPase.
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PMID:Calmodulin increases Ca-dependent inhibition of the Na,K-ATPase in human red blood cells. 131 6

A membrane fraction, enriched in Cl- channels, has been isolated from bovine tracheal epithelia and renal cortex homogenates by hydrophobic chromatography. The fraction (MPS) shows a 37-fold enrichment of Cl- channels over crude tracheal homogenates by net Cl- flux measurements. Alkaline phosphatase and Na(+)-K(+)-ATPase are not found in these membranes, suggesting that they are not apical or basolateral plasma membranes. Marker enzyme analysis for major subcellular membranes also proved negative. The MPS fraction exhibits a protein profile unlike that of other membrane fractions, with major proteins of 200 and 42 kDa, proteins of 30-35 kDa, and lesser amounts of other proteins. Reconstitution of MPS fractions from both trachea and kidney into planar lipid bilayers demonstrates the presence of a single type of anion channel. The current-voltage relationship of this channel is identical to that of the predominant anion channel observed in tracheal apical membranes under similar conditions (H. H. Valdivia, W. P. Dubinsky, and R. Coronado. Science Wash. DC 242: 1441-1444, 1988). In addition, the voltage dependence, selectivity sequence of Cl- > Br- > or = I-, and inhibition by low concentrations of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid correspond to those of the predominant apical membrane channel. Thus, although the MPS appear to be of subcellular origin, they may be functionally related to an apical membrane Cl- permeability.
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PMID:Isolation of a chloride channel-enriched membrane fraction from tracheal and renal epithelia. 132 48

This paper summarises results and conclusions from experiments with renal Na/K-ATPase, utilising proteolytic digestion to define minimal peptide structures involved in cation occlusion and chemical modification with dicyclohexylcarbodiimide (DCCD) to investigate the role of carboxyl groups and location of K (Rb) and/or Na binding residues. Extensive digestion with trypsin or non-selective proteases in the presence of Na or Rb and absence of divalent cations reveals an essential C-terminal 19Kd fragment of the alpha chain (N-terminal asn 830) and indicates that occlusion sites of Na or K ions must reside within transmembrane segments. The bulk of the beta chain is not involved. Kinetics of inactivation of Rb or Na occlusion and covalent labelling with DCCD indicate that each of two Rb(K) or Na sites contains a carboxyl group. The third Na site may contain only neutral ligating groups. One carboxyl group is located on the 19Kd fragment and the other on tryptic fragment of about 9Kd. When cyanogen bromide was used to digest labelled alpha chain, glu 953 was found to be labelled in a Rb-protectable fashion. In tryptic "19Kd-membranes", fragments containing all putative transmembrane segments of the alpha chain have been identified (i.e. 19, 10.9, 8.7 and 8.0 Kda respectively). The cation occlusion "cage" is apparently composed of ligating groups from different trans-membrane segments, including segments of the 19Kd fragment. Construction of models is hampered by the fact that the number of the transmembrane segments is still uncertain, particularly in the crucial C-terminal domain. Alternative ways of arranging the tryptic fragments across the membrane are discussed.
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PMID:Identification of the cation binding domain of Na/K-ATPase. 133 62

Fifteen specific inhibitors of DNA topoisomerases I and II were used to elucidate whether these enzymes participate in the excision repair of UV-induced DNA damage, monitoring DNA repair synthesis in confluent saponin-permeabilized human fibroblasts. To achieve a sufficient degree of accuracy dose--response experiments were performed, analysed by linear regression, and the concentrations at which repair activity was reduced to 50% were calculated and designated K50. Camptothecin, a specific inhibitor of topoisomerase I did not markedly diminish DNA repair synthesis. Similarly, when combined with topoisomerase II inhibitors [nalidixic acid, oxolinic acid, 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucop yra noside) (etoposide), 4'-demethylepipodophyllotoxin-thenylidene-beta-D-glucoside (teniposide), 1,4-dihydroxy-5,8-bis ((2-[(2-hydroxyethyl)amino]ethyl)amino)-9,10-anthracenedione (mitoxantrone), 5-(N-phenyl-carboxamido)-2-thiobarbituric acid (merbarone) or 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA)], it did not lower K50 values determined for topoisomerase II-specific drugs in separate experiments. The effects observed can be classified according to the mechanism of action the inhibitors exhibit. (i) Novobiocin and coumermycin, inhibitors of the ATPase subunit of topoisomerase II, completely reduced DNA repair synthesis. (ii) Inhibition of repair was also found for ethidium bromide, quinacrine and distamycin, drugs known to modify the DNA substrate by intercalation or binding to the DNA minor groove. (iii) Inhibitors acting through intercalation and, simultaneously, binding to the cleavable DNA-topoisomerase complex (m-AMSA, mitoxantrone, doxorubicin and daunorubicin) also suppressed reparative DNA synthesis. (iv) Only small effects were observed for etoposide, nalidixic acid and oxolinic acid, whereas teniposide caused marked inhibition of DNA repair synthesis. (v) Merbarone, a novel type of topoisomerase II inhibitor, blocked UV-induced DNA repair drastically. The results are best explained by assuming that in UV-irradiated human fibroblasts the 180 kDa form of topoisomerase II is the main target enzyme for inhibitors which suppressed DNA excision repair and that this isozyme is involved in steps preceding repair-specific DNA incision.
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PMID:The function of DNA topoisomerases in UV-induced DNA excision repair: studies with specific inhibitors in permeabilized human fibroblasts. 133 77

We have investigated the role, number, and identity of glutamate (or aspartate) residues involved in cation occlusion on Na+, K(+)-ATPase, using the carboxyl reagent N,N'-dicyclohexylcarbodiimide (DCCD). Extensive use is made of selectively trypsinized Na+,K(+)-ATPase--the so-called "19-kDa membranes"--containing a 19-kDa COOH-terminal, smaller (8-11 kDa) membrane-embedded fragments of the alpha chain, and a largely intact beta chain; these membranes have normal Rb+ and Na+ occlusion capacities. The 19-kDa peptide and a smaller (approximately 9 kDa) unidentified peptide(s) are labeled by [14C]DCCD in a Rb(+)-protectable fashion. Rb(+)-protected [14C]DCCD incorporation into the "19 kDa membranes" and into native Na+,K(+)-ATPase is linearly correlated with inactivation of Rb+ occlusion. Similar linear correlations are observed when Rb(+)-protected [14C]DCCD incorporation is measured by examination of labeling of 19-kDa peptide purified from "19-kDa membranes" or of alpha chain purified from native enzyme. Stoichiometries, estimated by extrapolation, are as follows: (for "19-kDa membranes") close to one DCCD per Rb+ site and one DCCD per 19-kDa peptide; and (for native enzyme) close to two DCCD per phosphoenzyme and two DCCD per alpha chain. We suggest that each of two K+ (or Na+) sites contains a carboxyl group, one located in the 19-kDa peptide and one elsewhere in the alpha chain. After cyanogen bromide digestion of purified, labeled alpha chain, or of 19-kDa peptide, a labeled fragment of apparent M(r) approximately 4 kDa was detected and was identified as that with NH2-terminal Lys-943. Rb(+)-protected [14C]DCCD incorporation was associated almost exclusively with Glu-953. We suggest that the cation occlusion "cage" consists of ligating groups donated by different trans-membrane segments and includes two carboxyl groups such as Glu-953 (and perhaps Glu-327) as well as neutral groups, in two K+ (or Na+) sites, but only neutral groups in the third Na+ site.
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PMID:Chemical modification of Glu-953 of the alpha chain of Na+,K(+)-ATPase associated with inactivation of cation occlusion. 135 83

The free Ca2+ concentrations in the nucleus ([Ca2+]n) and cytoplasm ([Ca2+]c) of cultured smooth muscle cells were estimated using the fluorescent dye indo-1 and the ACAS 570 confocal laser microscope. In resting DDT1MF2 smooth muscle cells [Ca2+]n was found to be lower than [Ca2+]c. Both values increased transiently in response to histamine (100 microM), but during this stimulation [Ca2+]n exceeded [Ca2+]c. Maximal increase of [Ca2+]n was observed in the center of the nucleus, and a maximal increase of [Ca2+]c was observed in the immediate vicinity of the plasma membrane. A similar response was obtained with other agonists, such as carbachol or ATP. Comparable results with ATP were obtained in cultured aorta cells. The differential rise of [Ca2+]n over [Ca2+]c in DDT1MF2 cells did not occur during either spontaneous release of Ca2+ or Ca2+ release induced by caffeine (7.5 mM). The differential rise during histamine stimulation was abolished by the presence of the intercalating substance ethidium bromide. Thapsigargin, a presumed specific inhibitor of the endoplasmic reticulum Ca(2+)-Mg(2+)-adenosine-triphosphatase, abolished the Ca2+ gradient between nucleus and cytosol at rest. During subsequent histamine stimulation the Ca2+ increase was largely blocked in both compartments and attained similar levels. We propose that the lower value of [Ca2+]n at rest is dependent on an active Ca2+ extrusion system. The differential rise of [Ca2+]n over [Ca2+]c during agonist stimulation can be explained by an influx of Ca2+ from perinuclear stores and/or by a release of intranuclear Ca2+ possibly mediated by a process dependent on the inositol lipid metabolism.
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PMID:Differences in regulation between nuclear and cytoplasmic Ca2+ in cultured smooth muscle cells. 138 89

The hydrophobic, photoactivatable probe TID [3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine] was used to label the plasma membrane H(+)-ATPase from Saccharomyces cerevisiae. The H(+)-ATPase accounted for 43% of the total label associated with plasma membrane protein and incorporated 0.3 mol of [125I]TID per mol of 100 kDa polypeptide. The H(+)-ATPase was purified by octyl glucoside extraction and glycerol gradient centrifugation, and was cleaved by either cyanogen bromide digestion or limited tryptic proteolysis to isolate labeled fragments. Cyanogen bromide digestion resulted in numerous labeled fragments of mass less than 21 kDa. Seven fragments suitable for microsequence analysis were obtained by electrotransfer to poly(vinylidene difluoride) membranes. Five different regions of amino-acid sequence were identified, including fragments predicted to encompass both membrane-spanning and cytoplasmic protein structure domains. Most of the labeling of the cytoplasmic domain was concentrated in a region comprising amino acids 347 to 529. This catalytic region contains the site of phosphorylation and was previously suggested to be hydrophobic in character (Goffeau, A. and De Meis, L. (1990) J. Biol. 265, 15503-15505). Complementary labeling information was obtained from an analysis of limited tryptic fragments enriched for hydrophobic character. Six principal labeled fragments, of 29.6, 20.6, 16, 13.1, 11.4 and 9.7 kDa, were obtained. These fragments were found to comprise most of the putative transmembrane region and a portion of the cytoplasmic region that overlapped with the highly labeled active site-containing cyanogen bromide fragment. Overall, the extensive labeling of protein structure domains known to lie outside the bilayer suggests that [125I]TID labeling patterns cannot be unambiguously interpreted for the purpose of discerning membrane-embedded protein structure domains. It is proposed that caution should be applied in the interpretation of [125I]TID labeling patterns of the yeast plasma membrane H(+)-ATPase and that new and diverse approaches should be developed to provide a more definitive topology model.
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PMID:Assessing hydrophobic regions of the plasma membrane H(+)-ATPase from Saccharomyces cerevisiae. 139 Aug 24

The equilibrium distribution of tetraphenylphosphonium bromide was used to measure the membrane potential in Leishmania donovani amastigotes and promastigotes and to investigate mechanisms underlying the maintenance of membrane potential. At pH 7.0, membrane potential ranges between -90 and -113 mV. Increasing the external concentrations of hydrogen or potassium ions decreased membrane potential as did treatments with carbonylcyanide chlorophenylhydrazone or valinomycin. These observations are consistent with a membrane potential set by hydrogen and potassium ion diffusion gradients. Anaerobiosis lowered membrane potential, suggesting the involvement of ATPase(s) in maintaining membrane potential. Membrane potential was insensitive to treatment with ouabain, demonstrating the absence of a Na+/K(+)-ATPase. Treatment with dicyclohexylcarbodiimide caused a temporary hyperpolarization of the membrane suggesting the participation of a proton ATPase in the maintenance of membrane potential. Determination of the membrane potential makes it possible to quantitate the total proton motive force which is the force for active transport across the parasite membrane.
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PMID:The plasma membrane electrical gradient (membrane potential) in Leishmania donovani promastigotes and amastigotes. 153 15

The improved reconstitution of the Mono Q-III fraction, a Cl(-)-translocating ATPase, isolated from Acetabularia acetabulum (Ikeda et al. (1990) Biochemistry 29, 2057-2065) into liposomes rendered transport properties of this enzyme clear. The liposomes were prepared by the reversed-phase method using egg lecithin and cholesterol in a molar ratio of 2:1 and the purified ATPase was incorporated into the liposomes by a dialysis for 3 h. About 80% of the ATPase was incorporated into the liposomes. The weight ratio of the enzyme to lipid was 1:400-600. A sigmoid curve was obtained when the Cl(-)-transport activity of the enzyme was plotted against Cl- concentration. Hill's plot afforded a half-substrate concentration [S]0.5 of 45 mM and a Hill's coefficient n of 2.33. Effects of Br- and F- on the Cl(-)-transport were also examined in the reconstituted system, both halide ions decreased the 36Cl- efflux significantly. These kinetic data are in good agreement with the electrophysiological data presented by Tittor et al. ((1983) J. Membr. Biol. 75, 129-139).
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PMID:Improvement of reconstitution of the Cl(-)-translocating ATPase isolated from Acetabularia acetabulum into liposomes and several anion pump characteristics. 153 90


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