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)

1. Purified pig kidney ATPase was incubated in 30--160 mM Tris-HCl with various monovalent cations. 130 mM LiCl stimulated a ouabain-sensitive ATP hydrolysis (about 5% of the maximal (Na+ + K) activity), whereas 160 mM Tris-HCl did not stimulate hydrolysis. Similar results were obtained with human red blood cell broken membranes. 2. In the absence of Na+ and with 130 mM LiCl, the ATPase activity as a function of KCl concentration showed an initial slight inhibition (50 micrometer KCl) followed by an activation (maximal at 0.2 mM KCl) and a further inhibition, which was total at mM KCl. In the absence of LiCl, the rate of hydrolysis was not affected by any of the KCl concentrations investigated. 3. The lithium-activation curve for ATPase activity in the absence of both Na+ and K+ had sigmoid characteristics. It also showed a marked dependence on the total LiCl + Tris-HCl concentration, being inhibited at high concentrations. This inhibition was more noticeable at low LiCl concentrations. 4. In the absence of Na+, 130 mM Li+ showed promoted phosphorylation of ATPase from 1 to 3 mM ATP in the presence of Mg2+. In enzyme treated with N-ethylmaleimide, the levels of phosphorylation in Li+-containing solutions, amounted to 40% of those in Na+- and up to 7 times of those in K+-containing solutions. 5. The total (Na+ + K+)-ATPase activity was markedly inhibited at high buffer concentrations (Tris-HCl, Imidazole-HCl and tetramethylammonium-HEPES gave similar results) in cases when either the concentration of Na+ or K+ (or both) was below saturation. On the other hand, the maximal (Na+ + K+)-ATPase activity was not affected (or very slightly) by the buffer concentration. 6. Under standard conditions (Tris-HCl + NaCl = 160 mM) the Na+-activation curve of Na+-ATPase had a steep rise between 0 and 2.5 mM, a fall between 2.5 and 20 mM and a further increase between 20 and 130 mM. With 30 mM Tris-HCl, the curve rose more steeply, inhibition was noticeable at 2.5 mM Na+ and was completed at 5 mM Na+. With Tris-HCl + NaCl = 280 mM, the amount of activation decreased and inhibition at intermediate Na+ concentrations was not detected.
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PMID:Activation by lithium ions of the inside sodium sites in (Na+ + K+)-ATPase. 21 14

Effects of lanthanum on Ca2+-ATPase, Mg2+-ATPase, Na+-K+-ATPase, and calcium binding activities were studied in rat heart sarcolemma. Ten to 100 micrometers lanthanum depressed significantly the Ca2+-ATPase activity and 50--200 micrometers lanthanum inhibited the calcium binding activity. Lineweaver-Burk plots of the Ca2+-ATPase activity showed that the inhibition by lanthanum was competitive with calcium concentration. Neither Mg2+-ATPase nor Na+-K+-ATPase activities were affected by lanthanum when the assay medium contained 1 mM EDTA; however, in the absence of EDTA, these enzyme activities were significantly decreased by 10--100 micrometers lanthanum. Rat hearts perfused with HEPES buffer containing 0.5 mM lanthanum showed electron-dense deposits restricted to the outer cell surface and the sarcolemma obtained from these hearts also had the deposits, indicating that the membrane fraction isolated by the hypotonic shock--LiBr treatment method is of sarcolemmal origin. The Ca2+-ATPase activity of the sarcolemma isolated from lanthanum-perfused hearts, unlike the Mg2+-ATPase, Na+-K+-ATPase, and calcium binding activities, was significantly less than the control value. From these observations it is suggested that lanthanum may influence calcium movement across the sarcolemma by affecting sarcolemmal ATPase and calcium binding activities.
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PMID:Effects of lanthanum on the heart sarcolemmal ATPase and calcium binding activities. 22 55

pH regulatory mechanisms in primary cultures of astrocytes from the cerebral cortex of neonatal audiogenic-seizure-susceptible DBA/2J (DBA) and genetically controlled C57BL/6J (C57) mice were studied with [14C]dimethyloxazolidine-2-4-dione (DMO) and [3H]-methyl-D-glucose (MDG). Effects of changing the concentration of Na+, K+, HCO3- or Cl- in medium, and/or of different transport blockers and metabolite inhibitor on intracellular pH (pHi) of cultured astrocytes were also studied. In nominal HCO3(-)-free HEPES-buffered Hanks' balanced salt solution (HEPES HBSS), when the pH of medium (pHo) was maintained at 7.4, the steady-state pHi of cultured astrocytes from DBA mice was 6.98 +/- 0.03, and that from C57 mice was 7.01 +/- 0.03. When the cells were incubated in HBSS containing 25 mM HCO3- and equilibrated with 5% CO2 (HCO3- HBSS, pHo = 7.4), pHi of both DBA and C57 astrocytes was approximately 0.1-0.15 pH units higher than that in HEPES HBSS. Reducing the pH or the Na+ concentration in media (pHo, [Na+]o) of either HEPES HBSS or HCO3- HBSS, pHi of both DBA and C57 astrocytes decreased markedly (0.25-0.45 pH units lower than the controls). The decrease in pHi was greater in HEPES HBSS than in HCO3- HBSS. Reducing the Cl- concentration ([Cl-]o) in either HEPES or HCO3- HBSS, pHi of astrocytes increased by 0.05-0.1 pH units. Increasing the K+ concentration ([K+]o) of or adding Ba2+ to the media increased the pHi of both DBA and C57 astrocytes accordingly. SITS, an anion transport inhibitor, decreased the pHi of both DBA and C57 astrocytes in HCO3- HBSS but not in HEPES HBSS. It enhanced the response of pHi to reduction in pHo. Amiloride, a Na(+)-H+ exchange inhibitor, decreased the pHi of both DBA and C57 astrocytes more in HEPES HBSS than in HCO3- HBSS. It enhanced the response of pHi to reduction in pHo and [Na+]o. Ouabain, an Na+,K(+)-ATPase inhibitor, decreased the pHi of cultured astrocytes in HEPES HBSS, but not in HCO3- HBSS. It also enhanced the response of pHi to changing pHo and [Na+]o in HEPES HBSS. Acetazolamide, a carbonic anhydrase inhibitor, decreased the pHi of astrocytes in both HEPES and HCO3- HBSS. Both bumetanide, an Na+,K+/Cl- cotransport blocker, and KCN, a metabolic inhibitor, produced no significant effect on the steady-state pHi or the response of pHi to changing ionic concentration in media in both DBA and C57 astrocytes.
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PMID:Studies on pH regulatory mechanisms in cultured astrocytes of DBA and C57 mice. 139 16

Potassium uptake and export in the resting conditions and in response to the phytohormone abscisic acid (ABA) were examined under voltage clamp in guard cells of Vicia faba L. In 0.1 mM external K+ (with 5 mM Ca2(+)-HEPES, pH 7.4) two distinct transport states could be identified based on the distribution of the free-running membrane voltage (VM) data in conjunction with the respective I-V and G-V relations. One state was dominated by passive diffusion (mean VM = -143 +/- 4 mV), the other (mean VM = -237 +/- 10 mV) exhibited an appreciable background of primary H+ transport activity. In the presence of pump activity the free-running membrane voltage was negative of the respective K+ equilibrium potential (EK+), in 3 and 10 mM external K+. In these cases VM was also negative of the activation voltage for the inward rectifying K+ current, thus creating a strong bias for passive K+ uptake through inward-rectifying K+ channels. In contrast, when pump activity was absent VM was situated positive of EK+ and cells revealed a bias for K+ efflux. Occasionally spontaneous voltage transitions were observed during which cells switched between the two states. Rapid depolarizations were induced in cells with significant pump activity upon adding 10 microM ABA to the medium. These depolarizations activated current through outward-rectifying K+ channels which was further amplified in ABA by a rise in the ensemble channel conductance. Current-voltage characteristics recorded before and during ABA treatments revealed concerted modulations in current passage through at least four distinct transport processes, results directly comparable to one previous study (Blatt, M.R., 1990, Planta 180:445) carried out with guard cells lacking detectable primary pump activity. Comparative analyses of guard cells in each case are consistent with depolarizations resulting from the activation of an inward-going, as yet unidentified current, rather than an ABA-induced fall in H(+)-ATPase output. Also observed in a number of cells was an inward-directed current which activated in ABA over a narrow range of voltages positive of -150 mV; this and additional features of the current suggest that it may reflect the ABA-dependent activation of an anion channel previously characterized in Vicia guard cell protoplasts, but rule out its function as the primary mechanism for initial depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Membrane transport in stomatal guard cells: the importance of voltage control. 153 80

We investigated the regulatory transport processes that maintain intracellular K+ homeostasis in cultured rat glomerular mesangial cells (MCs). Intracellular K+ concentration ([K+]i) of quiescent MCs, passages 3-8, grown to subconfluence on glass cover slips, was assessed by spectrofluorometry using the K(+)-sensitive dye, K(+)-binding benzofuran isophthalate (PBFI). Serum-starved MCs were incubated at 37 degrees C in 5 microM PBFI for 90 min. Excitation ratios of luminescences at 340 and 380 nm, measured at a constant emission at 500 nm, were used to determine [K+]i. Ionophores valinomycin and nigericin were used to clamp [K+]i to known [K+]o and thereby obtain an intracellular calibration of dye. Dependence of fluorescence ratio on [K+]i conformed to Michaelis-Menten behavior, with a Km of 113 mM (n = 40). PBFI retains its sensitivity to alterations in [K+]i with pH change (pHi from 6.5 to 7.5) but is relatively insensitive when intracellular Na+ is greater than 75 mM and cell osmolarity exceeds 500 mM. Normal resting [K+]i for all experiments was determined in MCs to be 102 +/- 7 mM (n = 81) in a HCO3(-)-free HEPES-buffered solution. When MCs were exposed to ouabain, [K+]i fell to 48 +/- 6 mM and did not recover, suggesting presence of Na(+)-K(+)-ATPase. When MCs were exposed to furosemide, [K+]i transiently declined to 58 +/- 11 mM, which was followed by a rapid recovery to near steady state, indicating additional presence of Na(+)-K(+)-Cl- cotransporter. Recovery was completely abolished when MCs were exposed to ouabain.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Regulation of intracellular potassium in mesangial cells: a fluorescence analysis using the dye, PBFI. 155 63

These studies were performed to determine the changes that occur in Na+/Ca2+ exchange activity in Alzheimer's disease (AD) brain tissues. Cerebral plasma membrane vesicles were purified by sucrose density gradient centrifugation from frozen postmortem hippocampal/temporal cortex tissue slices derived from age matched brains of normal, AD and non-Alzheimer dementia (NAD) origin (autopsy confirmed). Membrane marker assays (Na/K ATPase, muscarinic receptor, cytochrome c oxidase) revealed no change in membrane purity across different preparations. Thin-section electron microscopy revealed predominantly intact unilamellar vesicles. Vesicles were preincubated for 15 min (37 degrees C) in buffer containing 132 mM NaCl, 5 mM KCl, 1.3 mM MgCl2, 10 mM glucose and 10 mM HEPES (pH 7.4). Ca2+ uptake was initiated by diluting vesicles 20-fold with buffer containing either 132 mM NaCl or 132 mM choline chloride and 45CaCl2 then terminated by addition of 200 microM LaCl3 and rapid filtration. Ca2+ content increased rapidly at first and then maintained a steady plateau for up to 5 min. When the Ca2+ ionophore A23187 (10 microM) with 100 microM EGTA was added after 4 min, Ca2+ content was reduced to 10% of its original value. Ruthenium red (10 microM) had no effect on Ca2+ content. Na(+)-dependent Ca2+ uptake (Ca2+ content measured in choline chloride minus that measured in NaCl) was increased in AD brains as evidenced by both an increase in the initial rise in Ca2+ content and in elevated values of peak plateau Ca2+ content.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Na+/Ca2+ exchange activity is increased in Alzheimer's disease brain tissues. 164 56

Incubation of the murine macrophage tumour cell line PU5-1.8 in K+ (140 mM)-HEPES buffer induced depolarization of the membrane and the translocation of protein kinase C (PKC) to the subnuclear region. Membrane depolarization also induced an increase of intracellular free Ca2+ levels ([Ca2+]i) which was due to the Ca2+ influx. The amount of K(+)-mediated Ca2+ uptake was dependent on the Ca2+ concentration gradient as measured by indo-1 fluorescence and 45Ca2+ fluxes. The depolarization-mediated Ca2+ influx was suppressed by voltage sensitive Ca2+ channel blockers such as nifedipine and verapamil. Furthermore, in Na(+)-HEPES buffer, incubation of cells with a dihydropyridine agonist [3H]PN200-110 produced a dose-dependent saturable binding. On the other hand, short-term incubation of cells with phorbol 12-myristate 13-acetate (PMA) abolished the early phase of 45Ca2+ influx and the rise of indo-1 fluorescence. Depleting cells of PKC or incubating them with PKC inhibitors, H7 and sphingosine, enhanced the uptake of 45Ca2+ and the rise of indo-1 fluorescence. These observations suggest that membrane depolarization caused an activation of PKC and induced Ca2+ influx through the activation of dihydropyridine-sensitive, voltage-operated Ca2+ channels. Data also show that PKC may act as a negative modulator in controlling the Ca2+ response by closing the voltage-operated Ca2+ channel and/or by enhancing the Ca(2+)-ATPase activity during membrane depolarization in PU5-1.8 cells.
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PMID:Membrane depolarization induces protein kinase C translocation and voltage operated calcium channel opening in PU5-1.8 cells. Protein kinase C as a negative feedback modulator for calcium signalling. 165 33

The effect of extracellular pH (pHo) on the duration of calcium-dependent chloride currents (ICl(Ca] was studied in voltage clamped AtT-20 pituitary cells. ICl(Ca) was activated by Ca2+ influx through plasma membrane Ca2+ channels, which were opened by step depolarization to voltages between -20 and +60 mV. Increasing pHo from 7.3 to 8.0 reversibly prolonged ICl(Ca) tail currents in perforated patch recordings from cells bathed in both Na(+)-containing and Na(+)-free solutions. This prolongation was prevented in standard whole cell recordings when the pipette solution contained 0.5 mM EGTA. The effects of raised pHo were not due to alteration of intracellular pH, since tail current prolongation still occurred when intracellular pH was buffered at 7.3 with 80 mM HEPES. The prolongation of ICl(Ca) at pHo 8 could not be accounted for by a direct action on Ca2+ channels, since tail currents were prolonged when pHo was changed rapidly during the tail current, after all Ca2+ channels were closed. The effects of increasing pHo on ICl(Ca) also could not be explained by a direct action on Cl- channels, since changing to pHo 8 did not prolong Cl- tail currents when intracellular Ca2+ concentration [( Ca2+]i) was fixed by EGTA in whole cell recordings. Raising pHo did, however, prolong depolarization-evoked [Ca2+]i transients, measured directly with the Ca2+ indicator dye, fura-2. Taken together, these data demonstrate the presence of a Na(+)-independent, pHo-sensitive mechanism for reduction of [Ca2+]i after influx through Ca2+ channels. This mechanism is associated with the plasma membrane, and is active on a time scale that is relevant to the duration of single action potentials in these cells. We suggest that this mechanism is the plasma membrane Ca2+ ATPase.
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PMID:A [Na+]o-independent, pHo-dependent mechanism for reduction of intracellular [Ca2+] after influx through Ca2+ channels in mouse pituitary cells. 166 85

The thermodynamic efficiency of the calmodulin-activated form of the Ca2+-pumping ATPase of the bovine cardiac sarcolemma (SL) was evaluated in sealed vesicles under reversible conditions. The free internal Ca2+ concentration ([Ca2+]i) established in the SL vesicle lumen by action of the ATPase was determined as a function of the [ATP]/([ADP][Pi]) ratio for the following experimental conditions: 250 mM sucrose, 100 mM KCl, 0.1 mM Mg2+, 25 mM HEPES, 25 mM Tris, pH 7.40, at 37 degrees C, [Ca2+]o = 50 nM (1 mM Ca/EGTA buffer), 0.75 mM Mg-ATP, 0.1 mM Pi, variable [ADP]. Under these conditions, with the pump working near its Km of 64 nM, the [Ca2+]i achieved was less than or equal to 18 mM, decreasing with increasing [ADP] for [ADP] greater than or equal to 0.84 mM. A plot of the square of the [Ca2+]i/[Ca2+]o ratio against [ATP]/([ADP][Pi]) gave a straight line with a slope of 1.5 x 10(7) M. This was in agreement, within the experimental error, with the equilibrium constant for ATP hydrolysis under these conditions (1.09 x 10(7) M). These results demonstrate (1) tight coupling between Ca2+ transport and ATP hydrolysis with a stoichiometry of 2 Ca2+ moved per ATP split and (2) a low degree of passive leakage. Analysis at low [ADP] (less than 0.83 mM) showed the unexpected result that ADP increases the rate of the forward reaction of the pump. The maximal effect on the initial rate is a 96 +/- 5% increase, with an EC50 of approximately 0.4 mM (ADP). Similar but lesser stimulation was observed with CDP. The implications of the above results for the energetics of the pump and for its physiological function in the beating heart are discussed.
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PMID:The calmodulin-activated form of the Ca2(+)-pumping ATPase of the cardiac sarcolemmal membrane produces Ca2+ gradients with a thermodynamic efficiency of 100%. 213 38

The present investigation was designed to assess whether lens membrane permeability is affected by changes in levels of intracellular calcium. Lanthanum, an inhibitor of Ca-ATPase, affected an increase in the concentration of intracellular calcium (Cai) measured in cortical fiber cells. Preculture of lenses in lanthanum (1.0mM) caused an accumulation of 36Cl during subsequent culture at a rate three-fold higher than control lenses. Changes in calcium levels, however, were not responsible for the observed flux changes because a 40mV depolarization was observed to occur prior to a significant increase in calcium levels. The non-specific effects of lanthanum and other potential inhibitors of calcium transport were avoided by preculturing lenses in an ion-HEPES medium containing 20mM calcium chloride. In lenses with a six-fold increase in calcium levels there resulted only a 10% increase in 36Cl uptake over a 3 hr period. 86Rb efflux was also measured and the rate constant was unchanged compared to control lenses. Calcium accumulation did lead to a small (8mV) depolarization which may account for the small increase in chloride accumulation. By light microscopy, morphology of cortical lens fibers and the epithelium appeared unchanged in the calcium-loaded lens. The results provide little evidence that an increase in Cai leads to acute changes in lens membrane permeability.
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PMID:Effects of intracellular calcium on lens membrane permeability. 241 72


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