Water stress inhibits p-nitrophenyl phosphate hydrolysis activity of the plasma membrane H+-ATPase from soybean hypocotyls

2000 ◽  
Vol 27 (7) ◽  
pp. 717
Author(s):  
Quan-Sheng Qiu ◽  
Nan Zhang
Keyword(s):  
Plasma Membrane ◽  
Water Stress ◽  
Catalytic Mechanism ◽  
Gradient Centrifugation ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Phosphatase Domain ◽  
Nitrophenyl Phosphate ◽  
Peg Treatment ◽  
Pnpp Hydrolysis

The influence of water stress on ATP and p-nitrophenyl phosphate (PNPP) hydrolysis by plasma mem-brane ATPases was investigated using plasma membrane vesicles purified from soybean hypocotyls by the sucrose gradient centrifugation method. Results showed that ATPase activity was reduced after 10% polyethylene glycol (PEG) 6000 treatment for 12 h. Water stress also moved the optimal pH from 6.5 to 7.0. A significant decrease in PNPP hydrolysis was observed under PEG treatment. The Km for PNPP hydrolysis was shifted from 2.3042 0.0009 to 2.5048 0.0346 mmol L –1 . Moreover, PNPP hydrolysis was more sensitive to vanadate after PEG treatment, while inhibition of ATP hydrolysis by hydroxylamine was not affected. Our experimental results indicated that water stress changed the catalytic mechanism of the plasma membrane H + -ATPase through affecting the dephosphorylation process catalysed by its phosphatase domain.

scholarly journals Calcium transport mechanisms in basolateral plasma membrane-enriched vesicles from rat parotid gland

1985 ◽  
Vol 227 (1) ◽  
pp. 239-245 ◽  
Author(s):  
T Takuma ◽  
B L Kuyatt ◽  
B J Baum
Keyword(s):  
Plasma Membrane ◽  
Parotid Gland ◽  
Gradient Centrifugation ◽  
Membrane Vesicles ◽  
Transport Systems ◽  
Transport Mechanisms ◽  
Plasma Membrane Vesicles ◽  
Rat Parotid Gland ◽  
Basolateral Plasma Membrane ◽  
Rat Parotid

Ca2+ transport was studied by using basolateral plasma membrane vesicles from rat parotid gland prepared by a Percoll gradient centrifugation method. In these membrane vesicles, there were two Ca2+ transport systems; Na+/Ca2+ exchange and ATP-dependent Ca2+ transport. An outwardly directed Na+ gradient increased Ca2+ uptake. Ca2+ efflux from Ca2+-preloaded vesicles was stimulated by an inwardly directed Na+ gradient. However, Na+/Ca2+ exchange did not show any ‘uphill’ transport of Ca2+ against its own gradient. ATP-dependent Ca2+ transport exhibited ‘uphill’ transport. An inwardly directed Na+ gradient also decreased Ca2+ accumulation by ATP-dependent Ca2+ uptake. The inhibition of Ca2+ accumulation was proportional to the external Na+ level. Na+/Ca2+ exchange was inhibited by monensin, tetracaine and chlorpromazine, whereas ATP-dependent Ca2+ transport was inhibited by orthovanadate, tetracaine and chlorpromazine. Oligomycin had no effect on either system. These results suggest that in the parotid gland cellular free Ca2+ is extruded mainly by an ATP-dependent Ca2+ transport system, and Na+/Ca2+ exchange may modify the efficacy of that system.

scholarly journals A procedure for the rapid isolation from rat liver of plasma membrane vesicles exhibiting Ca2+-transport and Ca2+-ATPase activities

1984 ◽  
Vol 223 (3) ◽  
pp. 733-745 ◽  
Author(s):  
R J Epping ◽  
F L Bygrave
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Rat Liver ◽  
Gradient Centrifugation ◽  
Membrane Surface ◽  
Membrane Vesicles ◽  
Mitochondrial Fraction ◽  
Plasma Membrane Vesicles ◽  
Percoll Density Gradient Centrifugation ◽  
Percoll Density Gradient

A technique is described for the isolation of a plasma membrane-enriched preparation from a rat liver post-mitochondrial fraction by using discontinuous Percoll density-gradient centrifugation. The procedure is simple, of high reproducibility and yield and requires a total isolation time of only 90 min. The preparation consists almost exclusively of membrane vesicles and is enriched approx. 26-fold in plasma membrane-localized enzymes with minor contamination (less than 10%) with membranes derived mainly from the endoplasmic reticulum and Golgi apparatus. Approx. 20% of the fraction comprises tightly-sealed vesicles in the inverted orientation which are capable of accumulating calcium ions and exhibiting vanadate-insensitive Ca2+-ATPase activity. The properties of these activities, including insensitivity to vanadate, oxalate, and to p-chloromercuribenzoate as well as a lack of requirement for added Mg2+, contrast markedly with the reported properties of Ca2+ transport by the endoplasmic reticulum isolated from rat liver. The technique may have wide application in the study of plasma membrane-associated activities in rat liver, particularly in relation to sinusoidal membrane surface-related events.

Hexose transport in plasma membrane vesicles of rat myoblast L6

1984 ◽  
Vol 62 (11) ◽  
pp. 1217-1227 ◽  
Author(s):  
Matthias O. Cheung ◽  
Theodore C. Y. Lo
Keyword(s):  
Plasma Membrane ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
Electrical Potential ◽  
Membrane Vesicles ◽  
Hexose Transport ◽  
Plasma Membrane Vesicles ◽  
Transport Studies ◽  
Ethanesulfonic Acid ◽  
Flow Dialysis

To determine the molecular mechanism of hexose transport in rat myoblasts, transport studies were carried out with purified plasma membrane vesicles. Rat myoblasts were homogenized and fractionated by differential and sucrose gradient centrifugation. Six different fractions were obtained. Studies with marker enzymes revealed that two fractions (A and B) were composed of only plasma membrane. These two fractions differed considerably in their physical properties. Fraction A was composed of large multilaminated vesicles, with an intravesicular volume of 50 μL/mg protein, whereas fraction B was composed of membrane fragments and much smaller vesicles, with an intravesicular volume of 7 μL/mg protein. Based on the response of the ouabain-sensitive Na+,K+-ATPase activity to sodium dodecyl sulfate and ionophore treatments, it seemed likely that fraction A was composed of a significant amount of sealed right-side-out vesicles, whereas fraction B was composed of mainly membrane sheets or leaky vesicles. The initial rate of hexose influx into the membrane vesicles was determined by the flow dialysis technique. The optimal conditions for 2-deoxyglucose (2-DG) uptake into the plasma membrane vesicles were either 50 mM phosphate buffer or 10 mM 2-(N-2-hydroxyethylpiperazin-N′-yl)ethanesulfonic acid buffer at pH 7.0. In the presence of 500 μM 2-DG, the initial rates of 2-DG influx were 295 and 49 nmol/min per milligram protein for fractions A and B, respectively. In other words, after 1 min of incubation, the intravesicular concentration of 2-DG was around 6 mM, about 10 times the extravesicular concentration. D-Glucose was taken up to a similar extent (333 nmol/min per milligram protein), whereas L-glucose only equilibrated across the plasma membrane. Analysis of the fate of 2-DG revealed that the substrate was not phosphorylated upon incubation with the vesicles. Transport activity can be abolished either by disruption of the membrane vesicles or by reduction of the electrical potential across the membrane.

scholarly journals Plasma Membrane Vesicles from Mouse 15091A Tumor Cells: Agent of Platelet Aggregating Activity

1977 ◽  
Author(s):  
G.J. Gasic ◽  
J.L. Catafalmo ◽  
G.P. Gasic ◽  
S.J. Shattil ◽  
G.J. Stewart
Keyword(s):  
Plasma Membrane ◽  
Tumor Cells ◽  
Cell Transformation ◽  
Gradient Centrifugation ◽  
Amorphous Material ◽  
Membrane Vesicles ◽  
Sucrose Density Gradient ◽  
Plasma Membrane Vesicles ◽  
Sucrose Density Gradient Centrifugation ◽  
Artificial Plasma

We reported that cells from most tumors display platelet aggregating activity (PAA) in heparinized plasma and that this activity contributes to metastasis.Recently, we demonstrated that PAA can be used as a marker of cell transformation in virally infected rat cells. The material responsible for PAA is shed into culture medium. Characterization revealed a material which is particulate and sedimentable at 50,000x g for 60 min.; it contains proteins and lipids with a free cholesterol to phospholipid ratio of 0.556.Delipida-tion as well as complete solubilization abolished PAA.SDS-ME PAGE, 7.5% slab gels, revealed 20 bands.EM studies of 50,000x g pellets shed by 15091A cells indicated they contained numerous vesicles, some solid bodies, numerous free or vesicle associated small particles, and some amorphous material. Discontinuous sucrose density gradient centrifugation of the 50,000x g pellet yielded at the 1.07-1.17 g/cm3 interface a predominantly vesicular fraction which was the most active interfacial material. The vesicles, visible with phase contrast microscopy, resemble those produced by artificial plasma membrane vesiculation in various cell systems, including normal cells. Since PAA is only shown by transformed cells, vesicles from these must be different or much more numerous. Spontaneous vesiculation by tumor cells may be potentially important in understanding cell transformation and tumor metastases.

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.

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