scholarly journals Sterol Extraction from Isolated Plant Plasma Membrane Vesicles Affects H+-ATPase Activity and H+-Transport

Biomolecules ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1891
Author(s):  
Nikita K. Lapshin ◽  
Michail S. Piotrovskii ◽  
Marina S. Trofimova
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Membrane Vesicles ◽  
Hydrolytic Activity ◽  
Significant Inhibition ◽  
Plasma Membrane Vesicles ◽  
Kinetics Parameters ◽  
Hydrolytic Activities ◽  
Lipid Environment

Plasma membrane H+-ATPase is known to be detected in detergent-resistant sterol-enriched fractions, also called “raft” domains. Studies on H+-ATPase reconstituted in artificial or native membrane vesicles have shown both sterol-mediated stimulations and inhibitions of its activity. Here, using sealed isolated plasma membrane vesicles, we investigated the effects of sterol depletion in the presence of methyl-β-cyclodextrin (MβCD) on H+-ATPase activity. The rate of ATP-dependent ∆µH+ generation and the kinetic parameters of ATP hydrolysis were evaluated. We show that the relative sterols content in membrane vesicles decreased gradually after treatment with MβCD and reached approximately 40% of their initial level in 30 mM probe solution. However, changes in the hydrolytic and H+-transport activities of the enzyme were nonlinear. The extraction of up to 20% of the initial sterols was accompanied by strong stimulation of ATP-dependent H+-transport in comparison with the hydrolytic activity of enzymes. Further sterol depletion led to a significant inhibition of active proton transport with an increase in passive H+-leakage. The solubilization of control and sterol-depleted vesicles in the presence of dodecyl maltoside negated the differences in the kinetics parameters of ATP hydrolysis, and all samples demonstrated maximal hydrolytic activities. The mechanisms behind the sensitivity of ATP-dependent H+-transport to sterols in the lipid environment of plasma membrane H+-ATPase are discussed.

The influence of extracellular-side Ca2+ on the activity of the plasma membrane H+-ATPase from wheat roots

1998 ◽  
Vol 25 (8) ◽  
pp. 923 ◽  
Author(s):  
Quan-Sheng Qiu ◽  
Xue-Feng Su
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Gradient Centrifugation ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Two Phase ◽  
Wheat Roots ◽  
Hydrolytic Activities ◽  
Discontinuous Sucrose Gradient ◽  
Extracellular Side

Plasma membrane vesicles were purified from wheat roots by discontinuous sucrose gradient centrifugation and two-phase partitioning methods. The influence of extracellular-side Ca2+ on the activity of the plasma membrane H+ -ATPase from wheat roots was studied. The results showed that the ATP hydrolytic activities of the plasma membrane H+ -ATPase were inhibited by the cytoplasmic-side Ca2+. Within 0~200 µmol/L the ATPase activity decreased gradually with the increase in Ca2+ concentration; the ATPase activity was inhibited by 40% when Ca2+ concentration was 1000 µmol/L. However, the ATP hydrolytic activities were recovered by the presence of extracellular-side Ca2+. Results showed that the ATPase activities were increased with the increase in extracellular-side Ca2+; when the extracellular-side Ca2+ was 1000 µmol/L, the ATPase activity was recovered by 87.5%. Further studies found that the extracellular-side Ca2+ increased the DPH polarisation and decreased the MC540 fluorescence intensity, showing that membrane fluidity was decreased and membrane stacking was increased by the external Ca2+. The above results suggested that the plasma membrane H+ -ATPase could be regulated by the extracellular side Ca2+ through affecting the plasma membrane physical states.

Effect of antimicrobial peptides on ATPase activity and proton pumping in plasma membrane vesicles obtained from mycobacteria

Peptides ◽  
2012 ◽  
Vol 36 (1) ◽  
pp. 121-128 ◽  
Author(s):  
Paola Santos ◽  
Aldemar Gordillo ◽  
Luis Osses ◽  
Luz-Mary Salazar ◽  
Carlos-Yesid Soto
Keyword(s):  
Plasma Membrane ◽  
Antimicrobial Peptides ◽  
Atpase Activity ◽  
Membrane Vesicles ◽  
Proton Pumping ◽  
Plasma Membrane Vesicles

scholarly journals Characteristics of the Ca2+ pump and Ca2+-ATPase in the plasma membrane of rat myometrium

1988 ◽  
Vol 252 (1) ◽  
pp. 215-220 ◽  
Author(s):  
A Enyedi ◽  
J Minami ◽  
A J Caride ◽  
J T Penniston
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Calcium Uptake ◽  
Limited Proteolysis ◽  
Membrane Vesicles ◽  
Calcium Pump ◽  
Ionophore A23187 ◽  
Plasma Membrane Vesicles ◽  
Active Calcium ◽  
Stimulation Of

A plasma membrane-enriched fraction from rat myometrium shows ATP-Mg2+-dependent active calcium uptake which is independent of the presence of oxalate and is abolished by the Ca2+ ionophore A23187. Ca2+ loaded into vesicles via the ATP-dependent Ca2+ uptake was released by extravesicular Na+. This showed that the Na+/Ca2+ exchange and the Ca2+ uptake were both occurring in plasma membrane vesicles. In a medium containing KCl, vanadate readily inhibited the Ca2+ uptake (K1/2 5 microM); when sucrose replaced KCl, 400 microM-vanadate was required for half inhibition. Only a slight stimulation of the calcium pump by calmodulin was observed in untreated membrane vesicles. Extraction of endogenous calmodulin from the membranes by EGTA decreased the activity and Ca2+ affinity of the calcium pump; both activity and affinity were fully restored by adding back calmodulin or by limited proteolysis. A monoclonal antibody (JA3) directed against the human erythrocyte Ca2+ pump reacted with the 140 kDa Ca2+-pump protein of the myometrial plasma membrane. The Ca2+-ATPase activity of these membranes is not specific for ATP, and is not inhibited by mercurial agents, whereas Ca2+ uptake has the opposite properties. Ca2+-ATPase activity is also over 100 times that of calcium transport; it appears that the ATPase responsible for transport is largely masked by the presence of another Ca2+-ATPase of unknown function. Measurements of total Ca2+-ATPase activity are, therefore, probably not directly relevant to the question of intracellular Ca2+ control.

scholarly journals ATP-dependent transport of glutathione conjugate of 7β,8α-dihydroxy-9α,10α-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene in murine hepatic canalicular plasma membrane vesicles

1998 ◽  
Vol 332 (3) ◽  
pp. 799-805 ◽  
Author(s):  
Sanjay K. SRIVASTAVA ◽  
Xun HU ◽  
Hong XIA ◽  
Richard J. BLEICHER ◽  
Howard A. ZAREN ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Atp Hydrolysis ◽  
Membrane Vesicles ◽  
Competitive Inhibitor ◽  
Dependent Manner ◽  
Plasma Membrane Vesicles ◽  
High Affinity ◽  
Dependent Transport ◽  
Detoxification Pathway ◽  
Stimulation Of

Glutathione (GSH) S-transferases (GSTs) have an important role in the detoxification of (+)-anti-7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE], which is the ultimate carcinogen of benzo[a]pyrene. However, the fate and/or biological activity of the GSH conjugate of (+)-anti-BPDE [(-)-anti-BPD-SG] is not known. We now report that (-)-anti-BPD-SG is a competitive inhibitor (Ki 19 µM) of Pi-class isoenzyme mGSTP1-1, which among murine hepatic GSTs is most efficient in the GSH conjugation of (+)-anti-BPDE. Thus the inhibition of mGSTP1-1 activity by (-)-anti-BPD-SG might interfere with the GST-catalysed GSH conjugation of (+)-anti-BPDE unless one or more mechanisms exist for the removal of the conjugate. The results of the present study indicate that (-)-anti-BPD-SG is transported across canalicular liver plasma membrane (cLPM) in an ATP-dependent manner. The ATP-dependent transport of (-)-anti-[3H]BPD-SG followed Michaelis–Menten kinetics (Km 46 µM). The ATP dependence of the (-)-anti-BPD-SG transport was confirmed by measuring the stimulation of ATP hydrolysis (ATPase activity) by the conjugate in the presence of cLPM protein, which also followed Michaelis–Menten kinetics. In contrast, a kinetic analysis of ATP-dependent uptake of the model conjugate S-[3H](2,4-dinitrophenyl)-glutathione ([3H]DNP-SG) revealed the presence of a high-affinity and a low-affinity transport system in mouse cLPM, with apparent Km values of 18 and 500 µM respectively. The ATP-dependent transport of (-)-anti-BPD-SG was inhibited competitively by DNP-SG (Ki 1.65 µM). Likewise, (-)-anti-BPD-SG was found to be a potent competitive inhibitor of the high-affinity component of DNP-SG transport (Ki 6.3 µM). Our results suggest that GST-catalysed conjugation of (+)-anti-BPDE with GSH, coupled with ATP-dependent transport of the resultant conjugate across cLPM, might be the ultimate detoxification pathway for this carcinogen.

scholarly journals 131 PEROXIDATIVE CHALLENGE OF PLASMA MEMBRANE VESICLES FROM BELL PEPPER FRUIT LEADS TO DECREASED H+ATPase ACTIVITY

HortScience ◽  
1994 ◽  
Vol 29 (5) ◽  
pp. 447c-447
Author(s):  
Darlene M. Cowart ◽  
Robert L. Shewfelt
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Fruits And Vegetables ◽  
Chilling Injury ◽  
Membrane Vesicles ◽  
Thiobarbituric Acid ◽  
Plasma Membrane Vesicles ◽  
Water Control ◽  
Solubilized Enzyme

-Lipid peroxidation has been proposed as an important factor in chilling injury of susceptible fruits and vegetables. The effect of in vitro peroxidative challenge on H+ATPase activity in intact plasma membrane vesicles and solubilized enzyme was determined by incubation with (1) deionized water (control), (2) Fe3+-ascorbate, and (3) lipoxygenase (LOX) + phospholipase A2(PLA2) for 0, 30, and 60 min. Enzyme activity increased throughout the incubation period with no accumulation of thiobarbituric acid-reactive substances (TBA-RS) in the control, but vesicles challenged by the peroxidative systems showed significant increases in TBA-RS and decreases in membrane-bound H+ATPase activity. Greater losses in H+ATPase activity were observed in solubilized enzyme than in intact vesicles. The results indicate that loss of H+ATPase activity due to chemical modification of the protein rather than changes in membrane fluidity and suggest that modification is away from the active site.

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