scholarly journals Phorbol ester-sensitive phospholipase D is mainly localized in the endoplasmic reticulum of BHK cells

1996 ◽  
Vol 320 (3) ◽  
pp. 885-890 ◽  
Author(s):  
Christina DECKER ◽  
Maria Jesus MIRO OBRADORS ◽  
Daniel J. SILLENCE ◽  
David ALLAN
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Phospholipase D ◽  
Phorbol Ester ◽  
Membrane Vesicles ◽  
Sucrose Density Gradient ◽  
Subcellular Fractionation ◽  
Plasma Membrane Vesicles ◽  
Intact Cells ◽  
Site Of Action

The localization of phorbol ester-sensitive phospholipase D (PLD) in baby hamster kidney cells has been investigated by determining the subcellular distribution of the phosphatidylbutanol produced when the cells are incubated with phorbol 12-myristate 13-acetate and n-butanol. Results derived by isolation of plasma membrane vesicles from intact cells or by subcellular fractionation on a sucrose density gradient suggest the PLD is specific for phosphatidylcholine and its primary site of action is not the plasma membrane but the endoplasmic reticulum.

scholarly journals Isolation of a plasma-membrane fraction from gastric smooth muscle. Comparison of the calcium uptake with that in endoplasmic reticulum

1983 ◽  
Vol 210 (2) ◽  
pp. 315-322 ◽  
Author(s):  
L Raeymaekers ◽  
F Wuytack ◽  
J Eggermont ◽  
G De Schutter ◽  
R Casteels
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Muscle ◽  
Plasma Membrane ◽  
Membrane Fraction ◽  
Microsomal Fraction ◽  
Membrane Vesicles ◽  
Cholesterol Content ◽  
Sucrose Density Gradient ◽  
Plasma Membrane Vesicles ◽  
Plasma Membrane Fraction

1. A plasma-membrane fraction was isolated from the smooth muscle of the pig stomach by using differential and sucrose-density-gradient centrifugations. When the centrifugation was carried out after preloading the crude microsomal fraction with Ca2+ in the presence of oxalate, the contamination of the plasma-membrane fraction by endoplasmic reticulum was decreased and a fraction enriched in endoplasmic reticulum vesicles filled with calcium oxalate crystals was obtained. 2. The plasmalemmal and endoplasmic-reticulum membranes could be distinguished by differences in the activity of marker enzymes and in the cholesterol content and by their different permeability to oxalate and phosphate. Oxalate and phosphate stimulated the Ca2+ uptake in the endoplasmic reticulum much more than in the plasmalemmal vesicles. In the plasma-membrane vesicles 40 mM-phosphate was more effective for stimulating the Ca2+ uptake than was 5 mM-oxalate, but the reverse was seen in the endoplasmic reticulum. 3. The high cholesterol/phospholipid ratio of the crude microsomal fraction are of the majority of the vesicles present in the crude microsomal fraction are of plasmalemmal origin. 4. The Ca2+ pump of the plasmalemmal and endoplasmic-reticulum vesicles could be differentiated by their different sensitivities to calmodulin. However, the two Ca2+-transport ATPases did not differ by their sensitivity to vanadate nor by the energization of the Ca2+ transport by different nucleoside triphosphates.

scholarly journals Insertion of isolated insulin receptors into placental membrane vesicles

1992 ◽  
Vol 281 (2) ◽  
pp. 425-430 ◽  
Author(s):  
K Christiansen ◽  
J Carlsen
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Insulin Receptors ◽  
Membrane Vesicles ◽  
Insulin Binding ◽  
Scatchard Analysis ◽  
Plasma Membrane Vesicles ◽  
Intact Cells ◽  
Receptor Number ◽  
Triton X

Purified human insulin receptors were inserted into placental plasma-membrane vesicles by fusion of membranes with receptor-lysophosphatidylcholine micelles. Scatchard analysis of insulin binding showed that about 10-15% of the added receptors became inserted into the membrane. The receptor number could be increased about 3-fold, corresponding to approx. 5 pmol of receptor/mg of membrane protein. The receptors became firmly bound to the membrane, as they could not be removed by extensive wash. The insertion of exogenous receptors could be demonstrated by immunoblotting. The inserted insulin receptor had the same insulin-binding affinity as the isolated receptor and the endogenous receptor of the membrane. Insulin binding in the presence or absence of Triton X-100 revealed that more than 80% of the exogenous receptors had a right-side-out orientation. Function of the inserted receptors, as observed by insulin-stimulated autophosphorylation, could be demonstrated. About 80% of the added lysophospholipid, corresponding to approx. 160 nmol of lysophospholipid/mg of membrane protein, became integrated into the membrane and was partly metabolized to phospholipid and to non-esterified fatty acid. The method of insertion of isolated insulin receptors using the natural detergent, lysophospholipid, may be a method for insertion of receptors into intact cells, where the lysophospholipid, as in the plasma-membrane vesicles, will be acylated to phospholipid.

scholarly journals Biomembrane liquid–liquid phase separation and detergent resistance: a relationship strengthened

2009 ◽  
Vol 424 (2) ◽  
pp. e5-e6 ◽  
Author(s):  
David Holowka
Keyword(s):  
Plasma Membrane ◽  
Phase Separation ◽  
Liquid Phase ◽  
Lipid Rafts ◽  
Membrane Vesicles ◽  
Liquid Phase Separation ◽  
Plasma Membrane Vesicles ◽  
Intact Cells ◽  
Acyl Chains ◽  
Liquid Liquid Phase Separation

Since evidence first appeared for ‘detergent-resistant membranes’ in the early to mid-1990s, cell biologists from a wide spectrum of biological sciences have been intrigued by the functional relevance of this indication of membrane heterogeneity, commonly referred to as ‘lipid rafts’. Model membrane studies revealed that these lipid rafts are related to the more ordered liquid phase that forms in a ternary mixture of cholesterol with a phospholipid containing saturated acyl chains and one with unsaturated acyl chains. Giant plasma membrane vesicles that pinch off from cells undergo similar liquid–liquid phase separation as ternary model membranes, and have provided an experimental bridge between these and intact cells. The study by Levental et al. in this issue of the Biochemical Journal provides new insights into the relationship between liquid–liquid phase separation in these plasma membrane vesicles and detergent-resistance of cellular lipid rafts.

scholarly journals Characterization of Ca2+ fluxes in rat liver plasma-membrane vesicles

1988 ◽  
Vol 256 (1) ◽  
pp. 117-124 ◽  
Author(s):  
C Dargemont ◽  
M Hilly ◽  
M Claret ◽  
J P Mauger
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Uptake Rate ◽  
Ionic Composition ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Microsomal Preparation ◽  
Intact Cells ◽  
Inside Out

Inside-out plasma-membrane vesicles isolated from rat liver [Prpic, Green, Blackmore & Exton (1984) J. Biol. Chem. 259, 1382-1385] accumulated a substantial amount of 45Ca2+ when they were incubated in a medium whose ionic composition and pH mimicked those of cytosol and which contained MgATP. The Vmax of the initial 45Ca2+ uptake rate was 2.9 +/- 0.6 nmol/min per mg and the Km for Ca2+ was 0.50 +/- 0.08 microM. The ATP-dependent 45Ca2+ uptake by inside-out plasma-membrane vesicles was about 20 times more sensitive to saponin than was the ATP-dependent uptake by a microsomal preparation. The 45Ca2+ efflux from the inside-out vesicles, which is equivalent to the Ca2+ influx in intact cells, was increased when the free Ca2+ concentration in the medium was decreased. The Ca2+ antagonists La3+ and Co2+ inhibited the 45Ca2+ efflux from the vesicles. Neomycin stimulated the Ca2+ efflux in the presence of either a high or a low free Ca2+ concentration. These results confirm that polyvalent cations regulate Ca2+ fluxes through the plasma membrane.

scholarly journals myo-Inositol 1,4,5-trisphosphate mobilizes Ca2+ from isolated adipocyte endoplasmic reticulum but not from plasma membranes

1986 ◽  
Vol 236 (1) ◽  
pp. 37-44 ◽  
Author(s):  
D M Delfert ◽  
S Hill ◽  
H A Pershadsingh ◽  
W R Sherman ◽  
J M McDonald
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Plasma Membranes ◽  
Initial Rate ◽  
Membrane Vesicles ◽  
Heavy Fraction ◽  
Differential Centrifugation ◽  
Plasma Membrane Vesicles ◽  
Inositol 1,4,5 Trisphosphate ◽  
Uptake And Release

The effects of myo-inositol 1,4,5-trisphosphate (IP3) on Ca2+ uptake and release from isolated adipocyte endoplasmic reticulum and plasma membrane vesicles were investigated. Effects of IP3 were initially characterized using an endoplasmic reticulum preparation with cytosol present (S1-ER). Maximal and half-maximal effects of IP3 on Ca2+ release from S1-ER vesicles occurred at 20 microM- and 7 microM-IP3, respectively, in the presence of vanadate which prevents the re-uptake of released Ca2+ via the endoplasmic reticulum Ca2+ pump. At saturating IP3 concentrations, Ca2+ release in the presence of vanadate was 20% of the exchangeable Ca2+ pool. IP3-induced release of Ca2+ from S1-ER was dependent on extravesicular free Ca2+ concentration with maximal release occurring at 0.13 microM free Ca2+. At 20 microM-IP3 there was no effect on the initial rate of Ca2+ uptake by S1-ER. IP3 promoted Ca2+ release from isolated endoplasmic reticulum vesicles (cytosol not present) to a similar level as compared with S1-ER. Addition of cytosol to isolated endoplasmic reticulum vesicles did not affect IP3-induced Ca2+ release. The endoplasmic reticulum preparation was further fractionated into heavy and light vesicles by differential centrifugation. Interestingly, the heavy fraction, but not the light fraction, released Ca2+ when challenged with IP3. IP3 (20 microM) did not promote Ca2+ release from plasma membrane vesicles and had no effect on the (Ca2+ + Mg2+)-ATPase activity or on the initial rate of ATP-dependent Ca2+ uptake by these vesicles. These results support the concept that IP3 acts exclusively at the endoplasmic reticulum to promote Ca2+ release.

scholarly journals Delayed restoration of Mg2+ content and transport in liver cells following ethanol withdrawal

2009 ◽  
Vol 297 (4) ◽  
pp. G621-G631 ◽  
Author(s):  
Lisa M. Torres ◽  
Christie Cefaratti ◽  
Liliana Berti-Mattera ◽  
Andrea Romani
Keyword(s):  
Plasma Membrane ◽  
Membrane Vesicles ◽  
Isolated Hepatocytes ◽  
Liver Cells ◽  
Ethanol Withdrawal ◽  
Adrenergic Stimulation ◽  
Plasma Membrane Vesicles ◽  
Isolated Cells ◽  
Intact Cells

Liver cells from rats chronically fed a Lieber-De Carli diet for 3 wk presented a marked decreased in tissue Mg2+ content and an inability to extrude Mg2+ into the extracellular compartment upon stimulation with catecholamine, isoproterenol, or cell-permeant cAMP analogs. This defect in Mg2+ extrusion was observed in both intact cells and purified liver plasma membrane vesicles. Inhibition of adrenergic or cAMP-mediated Mg2+ extrusion was also observed in freshly isolated hepatocytes from control rats incubated acutely in vitro with varying doses of ethanol (EtOH) for 8 min. In this model, however, the defect in Mg2+ extrusion was observed in intact cells but not in plasma membrane vesicles. In the chronic model, upon removal of EtOH from the diet hepatic Mg2+ content and extrusion required ∼10 days to return to normal level both in isolated cells and plasma membrane vesicles. In hepatocytes acutely treated with EtOH for 8 min, more than 60 min were necessary for Mg2+ content and extrusion to recover and return to the level observed in EtOH-untreated cells. Taken together, these data suggest that in the acute model the defect in Mg2+ extrusion is the result of a limited refilling of the cellular compartment(s) from which Mg2+ is mobilized upon adrenergic stimulation rather than a mere defect in adrenergic cellular signaling. The chronic EtOH model, instead, presents a transient but selective defect of the Mg2+ extrusion mechanisms in addition to the limited refilling of the cellular compartments.

scholarly journals Compartmentalization of phosphatidylinositol 4,5-bisphosphate metabolism into plasma membrane liquid-ordered/raft domains

2021 ◽  
Vol 118 (9) ◽  
pp. e2025343118
Author(s):  
Jongyun Myeong ◽  
Cheon-Gyu Park ◽  
Byung-Chang Suh ◽  
Bertil Hille
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Resonance Energy ◽  
Membrane Vesicles ◽  
Membrane Lipid ◽  
Phosphoinositide Metabolism ◽  
Protein Markers ◽  
Plasma Membrane Vesicles ◽  
Intact Cells ◽  
Liquid Ordered

Possible segregation of plasma membrane (PM) phosphoinositide metabolism in membrane lipid domains is not fully understood. We exploited two differently lipidated peptide sequences, L10 and S15, to mark liquid-ordered, cholesterol-rich (Lo) and liquid-disordered, cholesterol-poor (Ld) domains of the PM, often called raft and nonraft domains, respectively. Imaging of the fluorescent labels verified that L10 segregated into cholesterol-rich Lo phases of cooled giant plasma-membrane vesicles (GPMVs), whereas S15 and the dye FAST DiI cosegregated into cholesterol-poor Ld phases. The fluorescent protein markers were used as Förster resonance energy transfer (FRET) pairs in intact cells. An increase of homologous FRET between L10 probes showed that depleting membrane cholesterol shrank Lo domains and enlarged Ld domains, whereas a decrease of L10 FRET showed that adding more cholesterol enlarged Lo and shrank Ld. Heterologous FRET signals between the lipid domain probes and phosphoinositide marker proteins suggested that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and phosphatidylinositol 4-phosphate (PtdIns4P) are present in both Lo and Ld domains. In kinetic analysis, muscarinic-receptor-activated phospholipase C (PLC) depleted PtdIns(4,5)P2 and PtdIns4P more rapidly and produced diacylglycerol (DAG) more rapidly in Lo than in Ld. Further, PtdIns(4,5)P2 was restored more rapidly in Lo than in Ld. Thus destruction and restoration of PtdIns(4,5)P2 are faster in Lo than in Ld. This suggests that Lo is enriched with both the receptor G protein/PLC pathway and the PtdIns/PI4-kinase/PtdIns4P pathway. The significant kinetic differences of lipid depletion and restoration also mean that exchange of lipids between these domains is much slower than free diffusion predicts.

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.

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