Ionic dependence of active Na-K transport: "clamping" of cellular Na+ with monensin

1987 ◽  
Vol 253 (1) ◽  
pp. F26-F33 ◽  
Author(s):  
R. S. Haber ◽  
T. A. Pressley ◽  
J. N. Loeb ◽  
F. Ismail-Beigi
Keyword(s):  
Atpase Activity ◽  
Liver Cell ◽  
Atp Hydrolysis ◽  
Hill Coefficient ◽  
Maximal Rate ◽  
Transport Capacity ◽  
Activation Curve ◽  
Intact Cells ◽  
K Transport ◽  
Ionophore Monensin

The Na+ ionophore monensin was used to study the Na+- and K+-dependence of ouabain-inhibitable 86Rb+ uptake in ARL 15 cells, a rat liver cell line. Graded concentrations of monensin rapidly induced incremental elevations of cellular Na+ that were stable for up to 2 h. In experiments in which cellular Na+ was thus “clamped” at various levels, the activation curve for ouabain-inhibitable 86Rb+ uptake as a function of intracellular Na+ was found to be steepest near basal Na+ levels (Hill coefficient approximately equal to 2.4), indicating that these cells can respond to relatively large changes in passive Na+ entry by increasing the race of Na-K pump function with only minimal increases in cellular Na+. Exposure of cells to monensin also permitted examination of the extracellular-K+ dependence of ouabain-inhibitable 86Rb+ uptake in the presence of saturating intracellular Na+ and yielded a Hill coefficient of approximately 1.5. The rate of ATP hydrolysis calculated from measurements of the maximal rate of ouabain-inhibitable 86Rb+ uptake in intact cells was similar to the enzymatic Vmax of the Na+-K+-ATPase in cell lysates, suggesting that the Na+-K+-ATPase activity in these broken-cell preparations closely reflects the functional transport capacity of the Na-K pump.

Na-K pump and Na-K-ATPase: disparity of their temperature sensitivity

1978 ◽  
Vol 235 (5) ◽  
pp. C159-C167 ◽  
Author(s):  
J. S. Willis ◽  
J. C. Ellory ◽  
J. H. Becker
Keyword(s):  
Temperature Sensitivity ◽  
Atp Hydrolysis ◽  
High Sensitivity ◽  
Temperature Characteristic ◽  
Ground Squirrels ◽  
Structural Reorganization ◽  
Intact Cells ◽  
Na Efflux ◽  
Resealed Ghosts ◽  
K Transport

As previously observed in red blood cells, ouabain-sensitive K influx of kidney cells grown in culture for 3 days was much less inhibited by cooling that Na-K-ATPase of the same cells. (At 5 degrees C K influx was 9.7% of that at 38 degrees C, Na-K-ATPase, 1--2%.) Resealed ghosts of erythrocytes of ground squirrels were made containing 24Na and ATP, and the Na efflux and ATP hydrolysis were measured simultaneously. Under these conditions there was no difference in the reduction of activity with cooling, and the amount of reduction was close to that of active K transport in intact cells. The high sensitivity to temperature, characteristic of broken membranes, could not be induced in intact cells or resealed ghosts by eliminating either the Na/K gradient or the ATP gradient nor by chelation of cellular and extracellular Ca. It could not be eliminated in broken membranes by protection with ATP or Mg. Structural reorganization of membrane during lysis may cause the increase in temperature sensitivity of Na-K-ATPase.

Mitochondrial injury: an early event in cisplatin toxicity to renal proximal tubules

1990 ◽  
Vol 258 (5) ◽  
pp. F1181-F1187 ◽  
Author(s):  
H. R. Brady ◽  
B. C. Kone ◽  
M. E. Stromski ◽  
M. L. Zeidel ◽  
G. Giebisch ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Proximal Tubule ◽  
Threshold Concentration ◽  
Early Event ◽  
Intact Cells ◽  
Mitochondrial Injury ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Early Effects ◽  
K Transport

Oxygen consumption (QO2) and net K+ transport were studied in rabbit proximal tubule suspensions to define the early effects of cisplatin on proximal tubule function. Cisplatin caused dose-dependent inhibition of QO2, which was delayed in onset. The concentration of cisplatin required for inhibition decreased as the duration of exposure was increased [40-min exposure, threshold concentration of 10(-4) M, inhibitor constant (Ki) of 10(-3) M; 4-h exposure, threshold concentration of 3 X 10(-5) M, Ki of 10(-4) M]. Both ouabain-sensitive and ouabain-insensitive QO2 were reduced, indicating inhibition of all adenosinetriphosphatases, including Na(+)- K(+)-ATPase activity. There was a parallel fall in ouabain-sensitive net K+ transport and cytosolic K+ content, confirming the latter observation. Na(+)-K(+)-ATPase activity was unchanged in cell membranes prepared by hypotonic lysis from cisplatin-treated tubules, indicating an indirect cytosol-dependent mechanism of enzyme inhibition. Nystatin-stimulated QO2 was reduced in cisplatin-treated tubules, excluding inhibition of Na+ entry as the mechanism of injury and suggesting mitochondrial injury. The latter was confirmed by measurement of carbonylcyanide-m-chlorophenylhydrazone (CCCP)-uncoupled QO2 in intact cells and ADP-stimulated (state 3) QO2 in digitonin-permeabilized tubules. Furthermore, by maximally stimulating mitochondrial respiration with CCCP and nystatin, it was possible to demonstrate mitochondrial injury at a time when basal QO2 and K+ transport were apparently normal. These data suggest that mitochondrial injury is a central event in cisplatin toxicity to the proximal tubule.

Interaction of iron regulatory protein-1 (IRP-1) with ATP/ADP maintains a non-IRE-binding state

2010 ◽  
Vol 430 (2) ◽  
pp. 315-324 ◽  
Author(s):  
Zvezdana Popovic ◽  
Douglas M. Templeton
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Binding Capacity ◽  
Regulatory Protein ◽  
Product Formation ◽  
Binding Activity ◽  
Hill Coefficient ◽  
Iron Regulatory Protein ◽  
Aconitase Activity ◽  
Iron Regulatory Protein 1

In its aconitase-inactive form, IRP-1 (iron regulatory protein-1)/cytosolic aconitase binds to the IRE (iron-responsive element) of several mRNAs to effect post-transcriptional regulation. We have shown previously that IRP-1 has ATPase activity and that binding of ATP suppresses the IRP-1/IRE interaction. In the present study, we characterize the binding activity further. Binding is observed with both [α-32P]ATP and [α-32P]ADP, but not with [γ-32P]ATP. Recombinant IRP-1 binds approximately two molecules of ATP, and positive co-operativity is observed with a Hill coefficient of 1.67±0.36 (EC50=44 μM) commencing at 1 μM ATP. Similar characteristics are observed with both apoprotein and the aconitase form. On binding, ATP is hydrolysed to ADP, and similar binding parameters and co-operativity are seen with ADP, suggesting that ATP hydrolysis is not rate limiting in product formation. The non-hydrolysable analogue AMP-PNP (adenosine 5′-[β,γ-imido]triphosphate) does not induce co-operativity. Upon incubation of IRP-1 with increasing concentrations of ATP or ADP, the protein migrates more slowly on agarose gel electrophoresis, and there is a shift in the CD spectrum. In this new state, adenosine nucleotide binding is competed for by other nucleotides (CTP, GTP and AMP-PNP), although ATP and ADP, but not the other nucleotides, partially stabilize the protein against spontaneous loss of aconitase activity when incubated at 37 °C. A mutant IRP-1(C437S) lacking aconitase activity shows only one ATP-binding site and lacks co-operativity. It has increased IRE-binding capacity and lower ATPase activity (Km=75±17 nmol/min per mg of protein) compared with the wild-type protein (Km=147±48 nmol/min per mg of protein). Under normal cellular conditions, it is predicted that ATP/ADP will maintain IRP-1 in a non-IRE-binding state.

Downmodulation of mitochondrial F0F1ATP synthase by diazoxide in cardiac myoblasts: a dual effect of the drug

2007 ◽  
Vol 292 (2) ◽  
pp. H820-H829 ◽  
Author(s):  
Marina Comelli ◽  
Giuliana Metelli ◽  
Irene Mavelli
Keyword(s):  
Atpase Activity ◽  
Atp Synthase ◽  
Transmembrane Potential ◽  
Atp Hydrolysis ◽  
Atp Synthesis ◽  
Mitochondrial Transmembrane Potential ◽  
Mitochondrial Atpase ◽  
Inhibitor Protein ◽  
Atp Content ◽  
Intact Cells

Similar to ischemic preconditioning, diazoxide was documented to elicit beneficial bioenergetic consequences linked to cardioprotection. Inhibition of ATPase activity of mitochondrial F0F1ATP synthase may have a role in such effect and may involve the natural inhibitor protein IF1. We recently documented, using purified enzyme and isolated mitochondrial membranes from beef heart, that diazoxide interacts with the F1sector of F0F1ATP synthase by promoting IF1binding and reversibly inhibiting ATP hydrolysis. Here we investigated the effects of diazoxide on the enzyme in cultured myoblasts. Specifically, embryonic heart-derived H9c2 cells were exposed to diazoxide and mitochondrial ATPase was assayed in conditions maintaining steady-state IF1binding (basal ATPase activity) or detaching bound IF1at alkaline pH. Mitochondrial transmembrane potential and uncoupling were also investigated, as well as ATP synthesis flux and ATP content. Diazoxide at a cardioprotective concentration (40 μM cell-associated concentration) transiently downmodulated basal ATPase activity, concomitant with mild mitochondria uncoupling and depolarization, without affecting ATP synthesis and ATP content. Alkaline stripping of IF1from F0F1ATP synthase was less in diazoxide-treated than in untreated cells. Pretreatment with glibenclamide prevented, together with mitochondria depolarization, inhibition of ATPase activity under basal but not under IF1-stripping conditions, indicating that diazoxide alters alkaline IF1release. Diazoxide inhibition of ATPase activity in IF1-stripping conditions was observed even when mitochondrial transmembrane potential was reduced by FCCP. The results suggest that diazoxide in a model of normoxic intact cells directly promotes binding of inhibitor protein IF1to F0F1ATP synthase and enhances IF1binding indirectly by mildly uncoupling and depolarizing mitochondria.

scholarly journals Positive co-operative activity and dimerization of the isolated ABC ATPase domain of HlyB from Escherichia coli

2005 ◽  
Vol 386 (3) ◽  
pp. 489-495 ◽  
Author(s):  
Houssain BENABDELHAK ◽  
Lutz SCHMITT ◽  
Carsten HORN ◽  
Kornelia JUMEL ◽  
Mark A. BLIGHT ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Atpase Activity ◽  
Analytical Ultracentrifugation ◽  
Atp Hydrolysis ◽  
Gel Filtration ◽  
Hill Coefficient ◽  
Type I ◽  
Atpase Domain ◽  
Important Species ◽  
Wide Range

The ATPase activity of the ABC (ATP-binding cassette) ATPase domain of the HlyB (haemolysin B) transporter is required for secretion of Escherichia coli haemolysin via the type I pathway. Although ABC transporters are generally presumed to function as dimers, the precise role of dimerization remains unclear. In the present study, we have analysed the HlyB ABC domain, purified separately from the membrane domain, with respect to its activity and capacity to form physically detectable dimers. The ATPase activity of the isolated ABC domain clearly demonstrated positive co-operativity, with a Hill coefficient of 1.7. Furthermore, the activity is (reversibly) inhibited by salt concentrations in the physiological range accompanied by proportionately decreased binding of 8-azido-ATP. Inhibition of activity with increasing salt concentration resulted in a change in flexibility as detected by intrinsic tryptophan fluorescence. Finally, ATPase activity was sensitive towards orthovanadate, with an IC50 of 16 μM, consistent with the presence of transient dimers during ATP hydrolysis. Nevertheless, over a wide range of protein or of NaCl or KCl concentrations, the ABC ATPase was only detected as a monomer, as measured by ultracentrifugation or gel filtration. In contrast, in the absence of salt, the sedimentation velocity determined by analytical ultracentrifugation suggested a rapid equilibrium between monomers and dimers. Small amounts of dimers, but apparently only when stabilized by 8-azido-ATP, were also detected by gel filtration, even in the presence of salt. These data are consistent with the fact that monomers can interact at least transiently and are the important species during ATP hydrolysis.

scholarly journals Synthesis of plasmalemmal glycoproteins in intestinal epithelial cells. Separation of Golgi membranes from villus and crypt cell surface membranes; glycosyltransferase activity of surface membrane

1978 ◽  
Vol 77 (3) ◽  
pp. 722-734 ◽  
Author(s):  
MM Weiser ◽  
MM Neumeier ◽  
A Quaroni ◽  
K Kirsch
Keyword(s):  
Cell Surface ◽  
Atpase Activity ◽  
Surface Membrane ◽  
Basal Membrane ◽  
Crypt Cell ◽  
Velocity Sedimentation ◽  
Intact Cells ◽  
Microvillus Membrane ◽  
Villus Cell ◽  
Crypt Cells

The relationship between Golgi and cell surface membranes of intestinal cells was studied. These membranes were isolated from intestinal crypt cells and villus cells. The villus cell membranes consisted of microvillus membrane, a Golgi-rich fraction, and two membrane fractions interpreted as representing lateral-basal membranes. The villus cell microvillus membrane was purified by previously published techniques while the other membranes were obtained from isolated cells by differential centrifugation and density gradient velocity sedimentation. The two membrane fractions obtained from villus cells and considered to be lateral-basal membranes were enriched for Na+,K+-ATPase activity, but one also showed enrichment in glycosyltransferase activity. The Golgi membrane fraction was enriched for glycosyltransferase activity and had low to absent Na+,K+-ATPase activity. Adenylate cyclase activity was present in all membrane fractions except the microvillus membrane but co-purified with Golgi rather than lateral-basal membranes. Electron microscopy showed that the Golgi fraction consisted of variably sized vesicles and cisternalike structures. The two lateral-basal membrane fractions showed only vesicles of smaller, more uniform size. After 125I labeling of isolated intact cells, radioactivity was found associated with the lateral-basal and microvillus membrane fractions and not with the Golgi fraction. Antibody prepared against lateral-basal membrane fractions reacted with the surface membrane of isolated villus cells. The membrane fractions from isolated crypt cells demonstrated that all had high glycosyltransferase activity. The data show that glycosyltransferase activity, in addition to its Golgi location, may be a significant property of the lateral-basal portion of the intestinal villus cell plasma membrane. Data obtained with crypt cells support earlier data and show that the crypt cell surface membrane possesses glycosyltransferase activity.

Tonoplast and plasma membrane ATPases from maize lines of high or low vigour

1992 ◽  
Vol 2 (2) ◽  
pp. 105-111 ◽  
Author(s):  
S. Sánchez-Nieto ◽  
R. Rodríguez-Sotres ◽  
P. González-Romo ◽  
I. Bernal-Lugo ◽  
M. Gavilanes-Ruíz
Keyword(s):  
Zea Mays ◽  
Plasma Membrane ◽  
Acid Phosphatase ◽  
Atpase Activity ◽  
Kinetic Analysis ◽  
Atp Hydrolysis ◽  
Membrane Atpases ◽  
Substrate Concentrations ◽  
Tonoplast Atpase ◽  
Specific Inhibitors

AbstractThe effectiveness of ATPase in germinated seed may play an important role in the vigour of germination. The activities of tonoplast and plasma membrane ATPases in two maize (Zea mays L.) lines with different vigour of germination were determined. ATP hydrolysis was measured in microsomal fractions from coleoptiles along with the responses to specific inhibitors for the plasma membrane, tonoplast and mitochondrial ATPases as well as for acid phosphatase. Nitrate-sensitive ATPase activity was 1.5–3.0 times lower in the low-vigour line than in the high-vigour line. Kinetic analysis of ATP hydrolysis at different substrate concentrations revealed the existence of two enzymes in the microsomal fractions of the two lines. The Vmax of enzyme 1 in the low-vigour line was a third of that in the high-vigour line. This enzyme was identified as the nitrate-sensitive or tonoplast ATPase on the basis of measurements of ATP hydrolysis in the presence of specific inhibitors at high (8.12mm) and low (0.77mm) ATP concentrations.

EAAT1 is involved in transport ofl-glutamate during differentiation of the Caco-2 cell line

2000 ◽  
Vol 279 (2) ◽  
pp. G366-G373 ◽  
Author(s):  
Agnès Mordrelle ◽  
Eric Jullian ◽  
Cyrille Costa ◽  
Estelle Cormet-Boyaka ◽  
Robert Benamouzig ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Line ◽  
Excitatory Amino Acid ◽  
Kinetic Studies ◽  
Glutamate Transport ◽  
Hill Coefficient ◽  
Amino Acid Transporters ◽  
Intestinal Epithelial ◽  
Transport Capacity ◽  
Excitatory Amino Acid Transporter

Little is known concerning the expression of amino acid transporters during intestinal epithelial cell differentiation. The transport mechanism ofl-glutamate and its regulation during the differentiation process were investigated using the human intestinal Caco-2 cell line. Kinetic studies demonstrated the presence of a single, high-affinity,d-aspartate-sensitive l-glutamate transport system in both confluent and fully differentiated Caco-2 cells. This transport was clearly Na+ dependent, with a Hill coefficient of 2.9 ± 0.3, suggesting a 3 Na+-to-1 glutamate stoichiometry and corresponding to the well-characterized XA,G − system. The excitatory amino acid transporter (EAAT)1 transcript was consistently expressed in the Caco-2 cell line, whereas the epithelial and neuronal EAAT3 transporter was barely detected. In contrast with systems B0 and y+, which have previously been reported to be downregulated when Caco-2 cells stop proliferating, l-glutamate transport capacity was found to increase steadily between day 8 and day 17. This increase was correlated with the level of EAAT1 mRNA, which might reflect an increase in EAAT1 gene transcription and/or stabilization of the EAAT1 transcript.

scholarly journals Identification of Rab6 as an N-ethylmaleimide-sensitive fusion protein-binding protein

2000 ◽  
Vol 352 (1) ◽  
pp. 165-173 ◽  
Author(s):  
Sang Yeul HAN ◽  
Dong Yoon PARK ◽  
Sang Dai PARK ◽  
Seung Hwan HONG
Keyword(s):  
Fusion Protein ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Polyclonal Antibodies ◽  
Binding Protein ◽  
Vesicular Transport ◽  
Brain Extract ◽  
Terminal Domain ◽  
Protein Receptor ◽  
And Function

In this study we show the interaction of N-ethylmaleimide-sensitive fusion protein (NSF) with a small GTP-binding protein, Rab6. NSF is an ATPase involved in the vesicular transport within eukaryotic cells. Using the yeast two-hybrid system, we have isolated new NSF-binding proteins from the rat lung cDNA library. One of them was Rab6, which is involved in the vesicular transport within the Golgi and trans-Golgi network as a Ras-like GTPase. We demonstrated that the N-terminal domain of NSF interacted with the C-terminal domain of Rab6, and these proteins were co-immunoprecipitated from the rat brain extract. This interaction was maintained preferentially in the presence of hydrolysable ATP. Recombinant NSF-His6 can also bind to C-terminal Rab6–glutathione S-transferase under the conditions to allow the ATP hydrolysis. Surprisingly, Rab6 stimulates the ATPase activity of NSF by approx. 2-fold as does α-soluble NSF attachment protein receptor. Anti-Rab6 polyclonal antibodies significantly inhibited the Rab6-stimulated ATPase activity of NSF. Furthermore, we found that Rab3 and Rab4 can also associate with NSF and stimulate its ATPase activity. Taken together, we propose a model in which Rab can form an ATP hydrolysis-regulated complex with NSF, and function as a signalling molecule to deliver the signal of vesicle fusion through the interaction with NSF.

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