Electrophysiological characterization of ATPases in native synaptic vesicles and synaptic plasma membranes

2010 ◽  
Vol 427 (1) ◽  
pp. 151-159 ◽  
Author(s):  
Petr Obrdlik ◽  
Kerstin Diekert ◽  
Natalie Watzke ◽  
Christine Keipert ◽  
Ulrich Pehl ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Synaptic Vesicles ◽  
Sucrose Gradient ◽  
Plasma Membranes ◽  
Specific Inhibitor ◽  
Chloride Conductance ◽  
Atpase Inhibitor ◽  
Electrical Measurements ◽  
Supported Membrane

Vesicular V-ATPase (V-type H+-ATPase) and the plasma membrane-bound Na+/K+-ATPase are essential for the cycling of neurotransmitters at the synapse, but direct functional studies on their action in native surroundings are limited due to the poor accessibility via standard electrophysiological equipment. We performed SSM (solid supported membrane)-based electrophysiological analyses of synaptic vesicles and plasma membranes prepared from rat brains by sucrose-gradient fractionation. Acidification experiments revealed V-ATPase activity in fractions containing the vesicles but not in the plasma membrane fractions. For the SSM-based electrical measurements, the ATPases were activated by ATP concentration jumps. In vesicles, ATP-induced currents were inhibited by the V-ATPase-specific inhibitor BafA1 (bafilomycin A1) and by DIDS (4,4′-di-isothiocyanostilbene-2,2′-disulfonate). In plasma membranes, the currents were inhibited by the Na+/K+-ATPase inhibitor digitoxigenin. The distribution of the V-ATPase- and Na+/K+-ATPase-specific currents correlated with the distribution of vesicles and plasma membranes in the sucrose gradient. V-ATPase-specific currents depended on ATP with a K0.5 of 51±7 μM and were inhibited by ADP in a negatively co-operative manner with an IC50 of 1.2±0.6 μM. Activation of V-ATPase had stimulating effects on the chloride conductance in the vesicles. Low micromolar concentrations of DIDS fully inhibited the V-ATPase activity, whereas the chloride conductance was only partially affected. In contrast, NPPB [5-nitro-2-(3-phenylpropylamino)-benzoic acid] inhibited the chloride conductance but not the V-ATPase. The results presented describe electrical characteristics of synaptic V-ATPase and Na+/K+-ATPase in their native surroundings, and demonstrate the feasibility of the method for electrophysiological studies of transport proteins in native intracellular compartments and plasma membranes.

Effects of bafilomycin A1 on cytosolic pH of sheep alveolar and peritoneal macrophages: evaluation of the pH-regulatory role of plasma membrane V-ATPases.

1995 ◽  
Vol 198 (8) ◽  
pp. 1711-1715 ◽  
Author(s):  
T A Heming ◽  
D L Traber ◽  
F Hinder ◽  
A Bidani
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Initial Rate ◽  
Cell Types ◽  
Peritoneal Macrophages ◽  
Bafilomycin A1 ◽  
Atpase Inhibitor ◽  
Cytosolic Ph ◽  
Phi Regulation

The role of plasma membrane V-ATPase activity in the regulation of cytosolic pH (pHi) was determined for resident alveolar and peritoneal macrophages (m theta) from sheep. Cytosolic pH was measured using 2',7'-biscarboxyethyl-5,6-carboxyfluorescein (BCECF). The baseline pHi of both cell types was sensitive to the specific V-ATPase inhibitor bafilomycin A1. Bafilomycin A1 caused a significant (approximately 0.2 pH units) and rapid (within seconds) decline in baseline pHi. Further, bafilomycin A1 slowed the initial rate of pHi recovery (dpHi/dt) from intracellular acid loads. Amiloride had no effects on baseline pHi, but reduced dpHi/dt (acid-loaded pHi nadir < 6.8) by approximately 35%. Recovery of pHi was abolished by co-treatment of m theta with bafilomycin A1 and amiloride. These data indicate that plasma membrane V-ATPase activity is a major determinant of pHi regulation in resident alveolar and peritoneal m theta from sheep. Sheep m theta also appear to possess a Na+/H+ exchanger. However, Na+/H+ exchange either is inactive or can be effectively masked by V-ATPase-mediated H+ extrusion at physiological pHi values.

The isolation of plasma membrane from protoplasts of soybean suspension cultures

1977 ◽  
Vol 24 (1) ◽  
pp. 295-310
Author(s):  
D.W. Galbraith ◽  
D.H. Northcote
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Membrane Fraction ◽  
Sucrose Gradient ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Membrane Systems ◽  
Enzymic Digestion ◽  
Nadh Cytochrome

A procedure for the isolation of plasma membranes from protoplasts of suspension-cultured soybean is described. Protoplasts were prepared by enzymic digestion of the cell wall and the plasma membrane was labelled with radioactive diazotized sulphanilic acid. The membrane systems from broken protoplasts were separated by continuous isopycnic sucrose gradient centrifugation. Radioactivity was localized in a band possessing a buoyant density of 1–14 g ml-1. The activities of NADPH- and NADH-cytochrome c reductase, fumarase, Mg2+-ATPase, IDPase and acid phosphodiesterase in the various regions of the density gradient were determined. A plasma membrane fraction was selected which was relatively uncontaminated with membranes derived from endoplasmic reticulum, tonoplasts and mitochondria. The results indicated that Mg2+-ATPase and possibly acid phosphodiesterase were associated with the plasma membrane.

scholarly journals Muscarinic acetylcholine receptor activation induces Ca2+ mobilization and Na+/K+-ATPase activity inhibition in eel enterocytes

2002 ◽  
Vol 173 (2) ◽  
pp. 325-334 ◽  
Author(s):  
A Muscella ◽  
S Greco ◽  
MG Elia ◽  
E Jimenez ◽  
C Storelli ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Muscarinic Acetylcholine Receptor ◽  
Specific Inhibitor ◽  
Fold Increase ◽  
Receptor Activation ◽  
Free Medium ◽  
Atpase Inhibitor ◽  
Extracellular Medium ◽  
Calphostin C ◽  
Dose Dependent

The effect of carbachol (Cch) on intracellular calcium concentration ([Ca2+]i) in eel enterocytes was examined using the fluorescent Ca2+ indicator fura-2. Cch caused a biphasic increase in [Ca2+]i, with an initial spike followed by a progressively decreasing level (over 6 min) to the initial, pre-stimulated, level. The effect of Cch was dose-dependent with a 7.5-fold increase in [Ca2+]i over basal level induced by the maximal dose of Cch (100 microM). In Ca2+-free/EGTA buffer the effect of Cch was less pronounced and the [Ca2+]i returned rapidly to basal levels. The increment of [Ca2+]i was dose-dependently attenuated in cells pre-treated with U73122, a specific inhibitor of phospholipase C, suggesting that the Cch-stimulated increment of [Ca2+]i required inositol triphosphate formation. In the presence of extracellular Ca2+, thapsigargin (TG), a specific microsomal Ca2+-ATPase inhibitor, caused a sustained rise in [Ca2+]i whereas in Ca2+-free medium the increase in [Ca2+]i was transient; in both cases, subsequent addition of Cch was without effect. When 2 mM CaCl2 were added to the cells stimulated with TG or with Cch in Ca2+-free medium, a rapid increase in [Ca2+]i was detected, corresponding to the capacitative Ca2+ entry. Thus, both TG and Cch depleted intracellular Ca2+ stores and stimulated influx of extracellular Ca2+ consistent with capacitative Ca2+ entry. K+ depolarization obtained with increasing concentrations of KCl in the extracellular medium induced a dose-related increase in [Ca2+]i which was blocked by 2 microM nifedipine, a non-specific L-type Ca2+ channel blocker. Nifedipine also changed significantly the height of the Ca2+ transient, and the rate of decrement to the pre-stimulated [Ca2+]i level, indicating that Ca2+ entry into enterocytes also occurs through an L-type voltage-dependent calcium channel pathway. We also show that isolated enterocytes stimulated with increasing Cch concentrations (0.1-1000 microM) showed a dose-dependent inhibition of the Na+/K+-ATPase activity. The threshold decrease was at 1 microM Cch; it reached a maximum at 100 microM (50.5% inhibition) and did not decrease further with the use of higher dose. The effect of Cch on Na+/K+-ATPase activity was dependent on both protein kinase C (PKC) and protein phosphatase calcineurin activation since the PKC inhibitor calphostin C abolished Cch effects, while the calcineurin inhibitor FK506 augmented Cch effect. Collectively, these data establish a functional pathway by which Cch can modulate the activity of the Na+/K+-ATPase through a PKC-dependent (calphostin C-sensitive) pathway and a calcineurin-dependent (FK506-sensitive) pathway.

scholarly journals Glucose-independent inhibition of yeast plasma-membrane H+-ATPase by calmodulin antagonists

1997 ◽  
Vol 322 (3) ◽  
pp. 823-828 ◽  
Author(s):  
Irma ROMERO ◽  
Ana M. MALDONADO ◽  
Pilar ERASO
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Atpase Activity ◽  
Plasma Membranes ◽  
Glucose Regulation ◽  
Wild Type ◽  
Calmodulin Antagonists ◽  
Dependent Protein Kinase ◽  
Yeast Plasma Membrane ◽  
Dependent Protein

Glucose metabolism causes activation of the yeast plasma-membrane H+-ATPase. The molecular mechanism of this regulation is not known, but it is probably mediated by phosphorylation of the enzyme. The involvement in this process of several kinases has been suggested but their actual role has not been proved. The physiological role of a calmodulin-dependent protein kinase in glucose-induced activation was investigated by studying the effect of specific calmodulin antagonists on the glucose-induced ATPase kinetic changes in wild-type and two mutant strains affected in the glucose regulation of the enzyme. Preincubation of the cells with calmidazolium or compound 48/80 impeded the increase in ATPase activity by reducing the Vmax of the enzyme without modifying the apparent affinity for ATP in the three strains. In one mutant, pma1-T912A, the putative calmodulin-dependent protein kinase-phosphorylatable Thr-912 was eliminated, and in the other, pma1-P536L, H+-ATPase was constitutively activated, suggesting that the antagonistic effect was not mediated by a calmodulin-dependent protein kinase and not related to glucose regulation. This was corroborated when the in vitroeffect of the calmodulin antagonists on H+-ATPase activity was tested. Purified plasma membranes from glucose-starved or glucose-fermenting cells from both pma1-P890X, another constitutively activated ATPase mutant, and wild-type strains were preincubated with calmidazolium or melittin. In all cases, ATP hydrolysis was inhibited with an IC50 of ≈1 μM. This inhibition was reversed by calmodulin. Analysis of the calmodulin-binding protein pattern in the plasma-membrane fraction eliminates ATPase as the calmodulin target protein. We conclude that H+-ATPase inhibition by calmodulin antagonists is mediated by an as yet unidentified calmodulin-dependent membrane protein.

Downregulation of surface sodium pumps by endocytosis during meiotic maturation of Xenopus laevis oocytes

1990 ◽  
Vol 258 (1) ◽  
pp. C179-C184 ◽  
Author(s):  
G. Schmalzing ◽  
P. Eckard ◽  
S. Kroner ◽  
H. Passow
Keyword(s):  
Plasma Membrane ◽  
Xenopus Laevis ◽  
Sucrose Gradient ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sodium Dodecyl ◽  
Meiotic Maturation ◽  
Xenopus Laevis Oocytes ◽  
Sodium Pumps

During meiotic maturation, plasma membranes of Xenopus laevis oocytes completely lose the capacity to transport Na and K and to bind ouabain. To explore whether the downregulation might be due to an internalization of the sodium pump molecules, the intracellular binding of ouabain was determined. Selective permeabilization of the plasma membrane of mature oocytes (eggs) by digitonin almost failed to disclose ouabain binding sites. However, when the eggs were additionally treated with 0.02% sodium dodecyl sulfate (SDS) to permeabilize inner membranes, all sodium pumps present before maturation were recovered. Phosphorylation by [gamma-32P]ATP combined with SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiography showed that sodium pumps were greatly reduced in isolated plasma membranes of eggs. According to sucrose gradient fractionation, maturation induced a shift of sodium pumps from the plasma membrane fraction to membranes of lower buoyant density with a protein composition different from that of the plasma membrane. Endocytosed sodium pumps identified on the sucrose gradient from [3H]ouabain bound to the cell surface before maturation could be phosphorylated with inorganic [32P]phosphate. The findings suggest that downregulation of sodium pumps during maturation is brought about by translocation of surface sodium pumps to an intracellular compartment, presumably endosomes. This contrasts the mechanism of downregulation of Na-dependent cotransport systems, the activities of which are reduced as a consequence of a maturation-induced depolarization of the membrane without a removal of the corresponding transporter from the plasma membrane.

scholarly journals Rhizosphere pH difference regulated by plasma membrane H+-ATPase is related to differential Al tolerance of two wheat cultivars

2011 ◽  
Vol 57 (No. 5) ◽  
pp. 201-206 ◽  
Author(s):  
Y. Yang ◽  
Q.L. Wang ◽  
M.J. Geng ◽  
Z.H. Guo ◽  
Z. Zhao
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Specific Inhibitor ◽  
Root Apex ◽  
Triticum Aestivum L ◽  
Rhizosphere Ph ◽  
Wheat Cultivars ◽  
Al Tolerance ◽  
Al Content ◽  
Tolerant Line

Aluminum (Al)-tolerance of different cultivars shows considerable differences. Elevation of rhizosphere pH is an external Al-resistant mechanism of plants. To elucidate the correlation between Al tolerance and the capacity of plants to modify the rhizosphere pH at different Al-tolerant levels, a comparative study on the wheat (Triticum aestivum L.) cultivars ET8 (Al-tolerant) and ES8 (Al-sensitive) was performed. Rhizosphere pH of ET8 was much higher than that of ES8 under the same treatment, significant correlations were obtained among all the data of rhizosphere pH and relative root elongation (R<sup>2</sup> = 0.9209**), or Al content in root apex (R<sup>2 </sup>= 0.9321**), which indicated that Al tolerance may be related to pH changes in the rhizosphere. The elevation of rhizosphere pH was inhibited by H<sup>+</sup>-ATPase specific inhibitor DCCD (dicylcohexylcarbodiimide, 25 mmol). Relative PM (plasma membrane) H<sup>+</sup>-ATPase activity of ET8 was significantly higher than that of ES8 under the same treatment. Significant correlation between all the data of relative PM H<sup>+</sup>-ATPase activity and rhizosphere pH (R<sup>2</sup> = 0.8319**) were obtained. Taken together, these results suggest that PM H<sup>+</sup>-ATPase was involved in regulating rhizosphere pH. Under Al stress, the Al-tolerant line showed a stronger capacity of up-regulating rhizosphere pH by PM H+-ATPase than the Al-sensitive line, which may explain the observed differences in Al tolerance between the two wheat cultivars.

Raft Localization and Nucleotidase Activity of CD39 Depends on Internal Proteolytic Cleavage

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3925-3925
Author(s):  
Kim E Olson ◽  
Joan HF Drosopoulos ◽  
Ashley E Olson ◽  
Marinus Johan Broekman ◽  
Aaron J Marcus
Keyword(s):  
Enzyme Activity ◽  
Plasma Membrane ◽  
Atpase Activity ◽  
Sucrose Gradient ◽  
Proteolytic Cleavage ◽  
High Density ◽  
Full Length ◽  
Low Density ◽  
293 Cells ◽  
Discontinuous Sucrose Gradient

Abstract We have previously shown that CD39 undergoes limited cleavage and that inhibition of proteolysis results in a decrease in ATPase activity. The reduction in enzymatic activity correlated with a decrease in the fraction of full-length CD39 present in active membrane raft-localized oligomeric complexes. We exploited N-and C-terminal VP16-and V5-tagged CD39, both transiently and stably expressed in 293 cells, to further elucidate the role of cleavage in the regulation of CD39 processing and activity. To characterize the complexes generated by cross-linking, N-terminal VP16-tagged and C-terminal V5-tagged CD39 were co-expressed in 293 cells. Following crosslinking of membranes with DTSSP and immunoprecipitation with anti-V5, DTT-cleaved species were visualized by Western Blot using VP16 antibody. Interestingly, both VP16-tagged full-length and N-terminal fragments (30 kDa) were immunoprecipitated by anti-V5. This indicates that both full-length CD39 and the N-terminal cleavage fragment are present in raft-localized complexes. The composition of raft-localized CD39 complexes was studied by separating membrane fractions on a discontinuous sucrose gradient using a non-detergent method. When overexpressed, CD39 and its C-terminal fragment distribute across the gradient as visualized by Western with anti-VP16. Importantly, specific activity (expressed as ATPase activity divided by total CD39 content) was 8 times greater in low-density raft-enriched fractions than in high density raft-free fractions. In addition, relative ADPase activity was higher in fractions containing a higher proportion of C-terminal CD39 relative to full-length CD39. Thus, CD39 forms oligomeric complexes and possesses optimal enzyme activity in lipid rafts. The relationship between CD39 cleavage, ATPase activity and raft localization was further studied in 293 cells transfected with C-or N-terminal VP16-tagged CD39. Subcellular fractionation on a discontinuous sucrose gradient yielded membrane fractions enriched in endoplasmic reticulum (ER), early endosomes (EE) and plasma membrane/Golgi (PM-Golgi). Importantly, the EE fraction contained both full-length and C-terminal (or N-terminal) CD39 at the same level as seen in the PM-Golgi fraction, suggesting that near 50% of CD39 resides in the EE compartment. Furthermore, EE-expressed CD39 exhibited an ATPase and ADPase activity equivalent to that seen in Golgi-PM fractions. This led us to examine effects of NH4Cl and bafilomycin (which block acidification of EE), and chloroquine (blocks EE maturation) on CD39 cleavage, activity and raft localization. Each treatment inhibited CD39 cleavage and correspondingly decreased ATPase activity. A shift of ~50% of full-length CD39 from raft fractions to high density membrane fractions was observed upon sucrose gradient fractionation following chloroquine treatment of cells transfected with N-terminal VP16 tagged CD39. This redistribution of CD39 in the membrane correlated with a 40% decrease in ATPase activity and a striking inhibition of CD39 cleavage. Here, at a lower level of expression than cited above, ATPase activity in low-density raft fractions was ~100-fold greater than in high density fractions. Thus, cleavage of a portion of CD39 molecules is required for both raft localization of full-length CD39 and optimal enzyme activity. Regulated proteolytic cleavage of CD39 would allow for rapid upregulation of CD39 activity in response to alterations in cell environment. This would occur via cycling of CD39 between plasma membrane and endosomal compartments, the proposed site of CD39 cleavage and assembly of fully active oligomeric complexes.

scholarly journals ATPases, ion exchangers and human sperm motility

Reproduction ◽  
2015 ◽  
Vol 149 (5) ◽  
pp. 475-484 ◽  
Author(s):  
Rubén D Peralta-Arias ◽  
Carmen Y Vívenes ◽  
María I Camejo ◽  
Sandy Piñero ◽  
Teresa Proverbio ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Sperm Motility ◽  
Plasma Membranes ◽  
Semen Analysis ◽  
Human Sperm ◽  
The Other ◽  
Computer Assisted ◽  
Acidic Ph ◽  
Α Subunit

Human sperm has several mechanisms to control its ionic milieu, such as the Na,K-ATPase (NKA), the Ca-ATPase of the plasma membrane (PMCA), the Na+/Ca2+-exchanger (NCX) and the Na+/H+-exchanger (NHE). On the other hand, the dynein-ATPase is the intracellular motor for sperm motility. In this work, we evaluated NKA, PMCA, NHE, NCX and dynein-ATPase activities in human sperm and investigated their correlation with sperm motility. Sperm motility was measured by Computer Assisted Semen Analysis. It was found that the NKA activity is inhibited by ouabain with twoKi(7.9×10−9and 9.8×10−5 M), which is consistent with the presence of two isoforms of α subunit of the NKA in the sperm plasma membranes (α1 and α4), being α4 more sensitive to ouabain. The decrease in NKA activity is associated with a reduction in sperm motility. In addition, sperm motility was evaluated in the presence of known inhibitors of NHE, PMCA and NCX, such as amiloride, eosin, and KB-R7943, respectively, as well as in the presence of nigericin after incubation with ouabain. Amiloride, eosin and KB-R7943 significantly reduced sperm motility. Nigericin reversed the effect of ouabain and amiloride on sperm motility. Dynein-ATPase activity was inhibited by acidic pH and micromolar concentrations of Ca2+. We explain our results in terms of inhibition of the dynein-ATPase in the presence of higher cytosolic H+and Ca2+, and therefore inhibition of sperm motility.

scholarly journals Characterization of the membrane proteins of rat liver lysosomes. Composition, enzyme activities and turnover

1982 ◽  
Vol 204 (2) ◽  
pp. 525-534 ◽  
Author(s):  
J Burnside ◽  
D L Schneider
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Atpase Activity ◽  
Enzyme Activities ◽  
Plasma Membranes ◽  
Lysosomal Membrane ◽  
Pulse Labelling ◽  
Polypeptide Composition ◽  
Lysosomal Membranes ◽  
Triton Wr 1339

Lysosomes prepared from the livers of untreated rats and from the livers of rats injected with either Triton WR-1339 or dextran yielded membranes that were similar in both polypeptide composition and activities of ATPase and acid 5'-nucleotidase. The administration of Triton WR-1339 (and dextran) resulted in an increase in ATPase activity of liver homogenates that was associated with a parallel increase in the ATPase activity of the lysosomal membrane. On the other hand, plasma membranes appear to be different from lysosomal membranes with respect to polypeptide composition and enzyme activities. The ATPase activity of lysosomal membranes is not affected by ouabain and suramin, inhibitors of the plasma-membrane ATPase. The plasma-membrane alkaline 5'-nucleotidase has little activity at acid pH. Pulse-labelling of lysosomal membranes with [3H]fucose and with [3H]- and [14C]-leucine occurred rapidly, faster than labelling of plasma membranes. The labelling kinetics indicate that lysosomal membranes may be assembled independently of plasma membranes. These data suggest that, in liver, little bulk transport of plasma membrane to lysosomes takes place, and lysosomal-membrane proteins may not be derived from those of plasma membranes.

Is there a Cl- pump?

1988 ◽  
Vol 255 (5) ◽  
pp. R677-R692
Author(s):  
G. A. Gerencser ◽  
J. F. White ◽  
D. Gradmann ◽  
S. L. Bonting
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Membrane Fraction ◽  
Plasma Membranes ◽  
Direct Evidence ◽  
Objective Evaluation ◽  
Chemical Processes ◽  
Transport Processes ◽  
Cl Transport ◽  
Active Ion Transport

Three universally accepted mechanisms of Cl- transport across plasma membranes exist and they are 1) anion-coupled antiport, 2) cation-coupled symport, and 3) coupling to primary active ion transport through electrical and/or chemical processes. No unequivocal direct evidence has been provided for primary active Cl- transport (Cl- pump) despite numerous reports of cellular Cl- -stimulated adenosinetriphosphatase (ATPases) and of Cl- transport that cannot be accounted for by the three well-documented Cl- transport processes. It has been demonstrated that Cl- -stimulated ATPase activity is localized to both mitochondrial and microsomal aspects of the cellular apparatus. However, one group ascribes microsomal localization of Cl- -stimulated ATPase activity to mitochondrial contamination of that membrane fraction. Therefore, no Cl- pump could ever exist naturally in any plasma membrane. The other group simply states that there is plasma membrane localization of Cl- -stimulated ATPase activity that could function as a Cl- pump. Both arguments are logically advanced and their conclusions are consistent with their respective premises. Resolution to the question Is there a Cl- pump? rests with each reader's critique and objective evaluation.

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