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 synthesis of Calcium Green C18, a lipophilic fluorescent calcium-sensitive dye, and its use as a monitor of Ca2+ efflux from cells is described. This indicator consists of a Calcium Green-1 molecule conjugated to a lipophilic 18-carbon alkyl chain which will intercalate into cell membranes. The Kd of the indicator for Ca2+ in aqueous solution (pH 7.2, 22 degrees C, ionic strength 0.1 M) is 0.23 +/- 0.04 microM and in the presence of liposomes is 0.062 +/- 0.007 microM. Due to its high negativity, the calcium chelating fluorophore faces the cell exterior, when loaded under a defined set of conditions. The dye was found largely on the surface of the cells when loaded at a concentration of 5 microM for 10 min at 37 degrees C. Five minutes after introduction of EGTA, 83-95% fluorescence disappeared, indicating that most of the fluorophore was on the cell surface. Photobleaching was minimal (3-13%). A confocal laser scanning microscope was used to detect and quantify fluorescence. Internalized dye was apparent in cells loaded for longer times (30-60 min) and in membrane-impaired cells, as shown by uptake of propidium iodide. Under defined confocal laser scanning microscope settings, a transient fluorescence at the periphery of approximately 30% of the cells was observed following 10(-8) M parathyroid hormone treatment, indicating the presence of outwardly directed calcium transport across the plasma membrane. Calcium efflux usually lasted 7-10 min, peaking at around 2-3 min. Changes in cell shape were also observed. Calcium efflux was shown to be sensitive to (a) 10 microM quercetin and 10 microM vanadate, partially specific inhibitors of plasma membrane Ca(2+)-ATPase, to (b) 0.1 mM trifluoperazine, an agent which renders calmodulin ineffective, and to (c) 10 mM neomycin sulfate, which blocks release of Ca2+ from intracellular stores. Thapsigargin (5 microM), an inhibitor of Ca(2+)-ATPase of the endoplasmic reticulum, prolonged fluorescence. These observations indicate that cell surface fluorescence was due to the capture of Ca2+ by Calcium Green C18 after Ca2+ had been translocated across osteoblast plasma membranes. Involvement of the plasma membrane Ca(2+)-ATPase, known to be present in osteoblasts in substantial amounts, is implicated.
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PMID:Characterization of calcium translocation across the plasma membrane of primary osteoblasts using a lipophilic calcium-sensitive fluorescent dye, calcium green C18. 767 32

Phospholipid molecules containing a cholate or hemisuccinylcholate moiety at the 2-position were synthesized as possible detergents for solubilization of membrane proteins. 1-Myristoleoyl-2-cholyl-sn-glycero-3- phosphatidylcholine ((C14:1,cholyl)PC) was found to solubilize sarcoplasmic reticulum vesicles at concentrations above its cmc of ca. 4 micrograms/ml. Effects of (C14:1,cholyl)PC on the activity of the Ca(2+)-ATPase of sarcoplasmic reticulum were complex, as for other detergents. High concentrations (0.2 mg/ml) of (C14:1,cholyl)PC were able to displace phospholipids from the lipid/protein interface of the ATPase. Although under these conditions the activity of the ATPase was low, the ATPase was not denatured since activity could be regained by displacement of (C14:1,cholyl)PC with the detergent C12E8. 1-oleoyl-2-cholyl-sn-glycero-3-phosphatidylcholine ((C18:1,cholyl)PC) was found to have a very low water solubility, but this could be increased by the introduction of a hemisuccinyl group to give 1-oleoyl-2-(3 alpha-hemisuccinyl)cholyl-sn-glycero-3-phosphatidylcholine ((C18:1,cholylCOOH)PC). This was able to solubilize and delipidate the Ca(2+)-ATPase; the ATPase was stable in (C18:1,cholylCOOH)PC for long periods of time.
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PMID:Synthesis of phospholipid-based detergents and their effects on the Ca(2+)-ATPase of sarcoplasmic reticulum. 769 91

The Ca(2+)-ATPase of skeletal-muscle sarcoplasmic reticulum, solubilized in monomeric from in C12E8, has been reconstituted by dialysis into sealed vesicles of dioleoyl phosphatidylcholine [di(C18:1)PC], dimyristoleoyl phosphatidylcholine [di(C14:1)PC], dinervonyl phosphatidylcholine [di(C24:1)PC] or dipalmitoyl phosphatidylcholine [di(C16:0)PC] in the gel phase, at a phospholipid/ATPase molar ratio of 10,000: 1. Cross-linking experiments show that ATPase molecules are present in these reconstituted vesicles as isolated monomeric species. ATPase activities for the reconstituted vesicles are about half of those for the ATPase reconstituted with the same lipid in unsealed membrane fragments, attributed to a close to random orientation for the ATPase molecules in the reconstituted vesicles. ATPase activities for the ATPase in reconstituted vesicles of di(C14:1)PC or di(C24:1)PC are less than in vesicles of di(C18:1)PC, and no activity could be detected for the ATPase in di(C16:0)PC in the gel phase. It is concluded that effects of lipids on the activity of the ATPase are independent of any changes in the state of aggregation of the ATPase. Inhibition of ATPase activity by spermine and by the hydrophilic domain of phospholamban are observed both for the unreconstituted ATPase and for the ATPase in reconstituted vesicles, so that inhibition is independent of any aggregation caused by these polycationic species. Stimulation of ATPase activity by jasmone is also observed both for the unreconstituted ATPase and for the ATPase in reconstituted vesicles, so that stimulation of the ATPase also does not follow from any change in the state of aggregation of the ATPase.
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PMID:Evidence that the effects of phospholipids on the activity of the Ca(2+)-ATPase do not involve aggregation. 775 84

On reconstitution of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum into bilayers of dimyristoleoylphosphatidylcholine [di(C14:1)PC] or dinervonylphosphatidylcholine [di(C24:1)PC] the stoichiometry of Ca2+ binding changes from the usual two Ca2+ ions bound per ATPase molecule to one Ca2+ ion bound per ATPase molecule. For the ATPase in di(C24:1)PC, removal of Ca2+ from the Ca(2+)-bound ATPase results in a decrease in tryptophan fluorescence intensity, as observed for the ATPase in dioleoylphosphatidylcholine [di(C18:1)PC]. For the ATPase in di(C14:1)PC removal of Ca2+ results in no change in tryptophan fluorescence intensity. In the presence of Mg2+, removal of Ca2+ from the ATPase in di(C18:1)PC or di(C24:1)PC results in a decrease in tryptophan fluorescence intensity, but for the ATPase in di(C14:1)PC this results in an increase in intensity. Fluorescence of the ATPase labelled with 4-nitrobenzo-2-oxa-1,3-diazole (NBD) is the same for the ATPase in di(C18:1)PC or di(C24:1)PC, but is markedly greater in di(C14:1)PC, consistent with a 4-fold increase in the E1/E2 equilibrium constant. Addition of Mg2+ to NBD-labelled ATPase in di(C18:1) PC or di(C24:1)PC results in an increase in NBD fluorescence, attributed to stronger binding of Mg2+ to the E1 than to the E2 conformation; addition of Mg2+ had no effect on the fluorescence of the NBD-labelled ATPase in di(C14:1)PC. In the absence of Ca2+, Mg2+ increased the tryptophan fluorescence of the ATPase in di(C14:1)PC, di(C18:3)PC or di(C24:1)PC, with the same binding-constant for Mg2+ in all three lipids. Addition of Mg2+ to the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin resulted in a decrease in fluorescence in di(C18:1)PC or di(C24:1)PC but had no effect in di(C14:1)PC. These effects are interpreted in terms of binding of Ca2+ at a single outer Ca2+ binding-site on the ATPase in di(C14:1)PC and di(C24:1)PC, in a conformation in which the inner site is occluded [in di(C14:1)PC] or modified in its affinity for Ca2+ [in di(C24:1)PC]. Thapsigargin binds to the ATPase, reducing its affinity for Ca2+ both in di(C14:1)PC and di(C24:1)PC.
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PMID:Characterization of the single Ca(2+)-binding site on the Ca(2+)-ATPase reconstituted with short- or long-chain phosphatidylcholines. 799 94

Escherichia coli transcription termination factor Rho (EC 3.6.1.3) releases nascent RNA from transcription complexes in a reaction which requires ATP hydrolysis. To understand the structure of the ATPase active site, we employed an analog of ATP, 8-azidoadenosine 5'-triphosphate (8-azido-ATP) as a photoaffinity labeling agent. 8-Azido-ATP interacts nearly normally with the active site of Rho. It binds to 3 sites per Rho hexamer with a 100 microM KD and is a substrate with a Vmax 5% that of ATP and a Km of 18 microM. Under UV irradiation, 8-azido-ATP makes covalent bonds with Rho, inactivating its ATPase. Rho is protected from this inactivation by the presence of ATP. We used [alpha-32P]8-azido-ATP to label the active site and identify residues involved in ATP binding. Labeled tryptic peptides of the modified Rho were purified by Fe(3+)-iminodiacetic acid affinity chromatography and reverse-phase C18 column high performance liquid chromatography. We identified a single peptide, Gly174-Lys184, that is labeled by 8-azido-ATP and protected from labeling in the presence of ATP. The modified amino acid is Lys181, whose conservative replacement by Gln181 gives rise to a poorly active enzyme (Dombroski, A. J., Brennan, C. A., Spear, P., and Platt, T. (1988a) J. Biol. Chem. 263, 18802-18809). Lys181 probably participates in binding the phosphoryl groups of ATP. Incorporation of one 8-azido-ATP per Rho hexamer is sufficient to cause inactivation, a result that indicates that the active sites of Rho interact in RNA-dependent ATP hydrolysis.
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PMID:8-Azido-ATP inactivation of Escherichia coli transcription termination factor Rho. Modification of one subunit inactivates the hexamer. 810 76

Phosphatidylcholines have been synthesized containing a cholesterol moiety at the 2-position of the glycerol backbone. Fluorescence quenching studies show that cholesterol-containing phosphatidylcholines can bind at the lipid-protein interface of the Ca(2+)-ATPase from skeletal muscle sarcoplasmic reticulum, with an affinity half that of dioleoylphosphatidylcholine. The ATPase activity measured for the ATPase reconstituted with the cholesterol-containing phosphatidylcholine containing an oleoyl fatty acyl chain, (C18:1, CHS)PC, is less than that measured for the ATPase reconstituted with dioleoylphosphatidylcholine. The activity measured for the ATPase reconstituted with the cholesterol-containing phosphatidylcholine containing a myristoleoyl fatty acyl chain, (C14:1, CHS)PC, is less than that measured in (C18:1,CHS)PC and is comparable to that measured in dimyristoleoylphosphatidylcholine (di(C14: 1)PC. The stoichiometry of Ca2+ binding to the ATPase is two Ca2+ ions bound per ATPase molecule in the native membrane or in (C18:1,CHS)PC, but one bound per ATPase molecule in di(C14:1)PC or (C14: 1,CHS)PC. Addition of cholesterol to the ATPase in di(C14:1)PC or (C14:1,CHS)PC increases the Ca2+ binding stoichiometry to the usual 2:1, but the binding stoichiometry remains 1:1 in mixtures of di(C14: 1)PC and (C14:1,CHS)PC. Removal of Ca2+ from the Ca(2+)-bound ATPase results in a decrease in tryptophan fluorescence intensity for the ATPase in the native membrane, but an increase in fluorescence intensity for the ATPase in di(C14:1)PC or (C14:1,CHS)PC. Addition of cholesterol to the ATPase in di(C14:1)PC or (C14:1,CHS)PC reverses this change. It is concluded that cholesterol linked to a phospholipid molecule can interact with the ATPase only at the lipid-protein interface.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Binding sites for cholesterol on Ca(2+)-ATPase studied by using a cholesterol-containing phospholipid. 816 59

Endogenous Na(+)-pump specific inhibitors are present in the plasma, urine, and tissues of humans and animals. To date, the source of these inhibitors has not been rigorously defined. In the present study, large amounts of several Na(+)-pump specific inhibitors have been demonstrated to exist in the urine of rats raised on a regular chow diet and tap water. All of the inhibitor levels have been found to increase 1.5-8-fold by the surgical preparation of reduced renal mass (RRM) and one-kidney, one-clip (IK, IC) hypertension. These urinary inhibitors, however, except for the ouabain-like inhibitor which eluted from a high performance liquid chromatography C18 column at the same retention time as [3H]ouabain, disappeared within a week after switching the diet from regular diet (number 5001, PMI Feeds, Inc.) to pure synthetic diet (number 5755). The urinary level of the ouabain-like inhibitor decreased to only one-half of the level in the control rat raised on a regular diet. Two of these inhibitors were purified from both urine and diet by a combination of Amberlite XAD-2 adsorption chromatography, reverse phase low pressure liquid chromatography, and several high performance liquid chromatographies. Reverse phase high performance liquid chromatography, liquid secondary ion and gas-liquid mass spectrometries, and proton nuclear magnetic resonance spectroscopy identified these inhibitors as a stereoisomer of convalloside, probably neoconvalloside, and a mono-rhamnoside of periplogenin or its stereoisomer. These cardiac glycosides exhibited inhibitory potencies comparable to ouabain against ouabain-displacement from Na+,K(+)-ATPase and against 86Rb uptake into human erythrocytes, and they also exhibited cross-reactivity to anti-ouabain antibodies and anti-digoxin antibodies. These results clearly demonstrate that the principal source of most of the inhibitors in rat urine is the diet. The results suggest that the ouabain-like inhibitor may be derived from an endogenous origin.
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PMID:Identification of two cardiac glycosides as Na(+)-pump inhibitors in rat urine and diet. 816

Circulating inhibitors of the transport enzyme, sodium-potassium-activated adenosine triphosphatase (Na(+)-K(+)-ATPase), have been shown to be of possible pathogenetic importance in the mechanism of essential hypertension. Although previous studies have demonstrated the presence of both high molecular weight (HMW) and low molecular weight (LMW) natriuretic plasma Na(+)-K(+)-ATPase inhibitors, no previous attempts have been made to ascertain whether HMW or LMW forms predominate in hypertension. In this study, plasma samples obtained from 26 patients with essential hypertension, 12 normotensive controls, and six normotensives with a family history of hypertension, were separated into HMW and LMW moieties by passage through a 1 kDa Amicon membrane. The LMW moiety was separated on C18 Sep-Pak cartridges, applying a 10% step-wise acetonitrile trifluoroacetic acid gradient. The HMW moiety was further separated on Sephadex G-75. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the fraction with inhibitory activity contained a distinct 12 kDa protein band, with staining intensity depending on the presence or absence of hypertension. Na(+)-K(+)-ATPase inhibitory activity was found in several LMW fractions, but differences between hypertensives and normotensives were observed in only one fraction (0.29 +/- 0.12 SD v 0.11 +/- .12 mumol/L ouabain equivalents, P < .01). Na(+)-K(+)-ATPase inhibitory activity in the HMW fraction was 38 x the inhibitory activity in the LMW fraction and was significantly increased in hypertensives as compared to normotensive controls (10.9 +/- 8.9 v 1.3 +/- 0.8 mumol/L ouabain equivalents, P < .01). Inhibitory activity in both HMW and LMW fractions correlated positively with mean blood pressure.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Predominance of high molecular weight plasma Na(+)-K(+)-ATPase inhibitor in essential hypertension. 821 31

A Na+,K(+)-ATPase inhibitor was purified from 88.6 l pig urine with a yield of approximately 10 micrograms. It inhibits the ouabain-sensitive uptake of 86Rb into human erythrocytes and the specific binding of ouabain to Na+,K(+)-ATPase. It also exhibits cross-reactivity to anti-ouabain serum. The purification procedure consisted of adsorption chromatography on an Amberlite XAD-2 column, preparative scale C18 low-pressure liquid chromatography (LPLC), and five steps of HPLC with five different types of reverse-phase columns. The dose dependence of the purified substance for the inhibition of ouabain-sensitive Na+,K(+)-ATPase activity and 86Rb uptake into human erythrocytes, and for the ouabain-displacing activity, paralleled those of ouabain, spanning two orders of magnitude in concentration range. However, the curve obtained from the cross-immunoreactivity with anti-ouabain serum did not parallel that of ouabain. The inhibitory potencies of the purified substance against the Na(+)-pump and ouabain-binding were diminished with increasing K+ concentration, exhibiting characteristics typical of cardiac glycosides. This substance had no effect on Ca(2+)-ATPase activity in human erythrocyte plasma membrane and skeletal-muscle sarcoplasmic reticulum, nor on Mg(2+)-ATPase activity. Acid treatment with 6 M HCl at 115 degrees C for less than 1 min destroyed approximately 82% of the inhibitory activity of the purified substance against Na(+)-pump activity. Alkaline treatment with 0.2 M NaOH at 23 degrees C for 2 h and heat treatment at 150 degrees C for 30 min partially destroyed the inhibitory activity. Boiling for 10 min and digestion by various enzymes did not affect the activity. Molecular mass was estimated to be 620 Da by gel-filtration column chromatography. Preliminary MS analysis suggested that the purified substance has a molecular mass of 625 Da. An 1H-NMR study revealed that this substance does not contain a tertiary methyl group. The results suggest that the purified Na+,K(+)-ATPase inhibitor is not a peptide and is distinct from any of the known cardiotonic steroids or various substances previously reported to exhibit Na+,K(+)-ATPase inhibitory activity. Thus, the purified substance may be a novel endogenous regulator of Na+,K(+)-ATPase.
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PMID:Isolation and characterization of an endogenous Na+,K(+)-ATPase-specific inhibitor from pig urine. 838 Oct 85

Numerous studies on the molecular basis of the mechanism of action of fatty acids have demonstrated their action in cell signaling and particularly on the regulation of cytosolic Ca2+ concentration. Stimulation of Jurkat T cells with CD3 monoclonal antibody results in an increase of intracellular calcium concentration, [Ca2+]i due both to a release of Ca2+ from intracellular stores and a Ca2+ influx. [Ca2+]i increase represents a dynamic balance between Ca2+ influx and efflux. Fatty acids, either saturated (C14:0), monounsaturated (C16:1), or polyunsaturated, belonging to the C18 and the C20 series induce a marked decrease of CD3-induced [Ca2+]i rise. This property of fatty acid is independent of the position of the carbon-carbon double bond but specific of the cis stereoisomeric form. Fatty acids does not block CD3-induced signals but greatly stimulates the Ca2+ extrusion process probably by activating the plasma membrane Ca(2+)-ATPase. This was documented by the observation that fatty acids, reduced to the same extent as [Ca2+]i, elicited either with CD3 monoclonal antibody the calcium ionophore ionomycin or the Ca(2+)-ATPase inhibitor thapsigargin.
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PMID:The inhibition by fatty acids of receptor-mediated calcium movements in Jurkat T-cells is due to increased calcium extrusion. 840 10


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