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.

Electroimmunochemical analysis of plasma membrane vesicles from Saccharomyces cerevisiae

1982 ◽  
Vol 60 (6) ◽  
pp. 659-667
Author(s):  
James H. Gerlach ◽  
Ole J. Bjerrum ◽  
Gerald H. Rank
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Concanavalin A ◽  
Periodic Acid ◽  
Membrane Vesicles ◽  
Free Form ◽  
Plasma Membrane Vesicles ◽  
Crossed Immunoelectrophoresis ◽  
Lentil Lectin ◽  
Triton X

Plasma membrane vesicles of Saccharomyces cerevisiae were extracted with 1% (w/v) Triton X-100 and the solubilized proteins examined by crossed immunoelectrophoresis using rabbit antibodies against the vesicles. Solubilization was shown to be nonselective and 23 immunoprecipitates were observed reproducibly.Four glycoproteins were identified by interaction with concanavalin A and lentil lectin, either immobilized on agarose beads in an intermediate gel or incorporated in the free form in the first dimension gel. One glycoprotein was stainable by the periodic acid – Schiff procedure. None of the glycoproteins had their origin in the cell wall.Five amphiphilic proteins were identified on the basis of charge-shift and hydrophobic interaction crossed immunoelectrophoresis as well as [14C]Triton X-100 and Sudan black B binding. Three of the amphiphilic proteins were also glycoproteins.Based on the carbohydrate content and amphiphilic properties of the proteins, purification schemes using concanavalin A-Sepharose and phenyl-Sepharose were proposed. Trial separations using 1-mL columns were monitored by fused rocket and crossed immunoelectrophoresis.

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 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 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 Diffusion of lipids and GPI-anchored proteins in actin-free plasma membrane vesicles measured by STED-FCS

2016 ◽  
Author(s):  
Falk Schneider ◽  
Mathias P Clausen ◽  
Dominic Waithe ◽  
Thomas Koller ◽  
Gunes Ozhan ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
High Mobility ◽  
Super Resolution ◽  
Membrane Vesicles ◽  
Correlation Spectroscopy ◽  
Diffusion Regime ◽  
Plasma Membrane Vesicles ◽  
Intact Cells ◽  
Actin Cortex ◽  
Hindered Diffusion

Diffusion and interaction dynamics of molecules at the plasma membrane play an important role in cellular signalling. These have been suggested to be strongly associated with the actin cytoskeleton. Here, we utilise super-resolution STED microscopy combined with fluorescence correlation spectroscopy (STED-FCS) to access the sub-diffraction diffusion regime of different fluorescent lipid analogues and GPI-anchored proteins (GPI-APs) in the cellular plasma membrane, and compare it to the diffusion regime of these molecules in cell-derived actin-free giant plasma membrane vesicles (GPMVs). We show that phospholipids and sphingomyelin, which undergo hindered diffusion in the live cell membrane, diffuse freely in the GPMVs. In contrast to sphingomyelin, which is transiently trapped on molecular-scale complexes in intact cells, diffusion of the ganglioside lipid GM1 suggests transient incorporation into nanodomains, which is less influenced by the actin cortex. Finally, our data on GPI-APs indicate two molecular pools in living cells, one pool showing high mobility with trapped and compartmentalized diffusion, and the other forming immobile clusters both of which disappear in GPMVs. Our data underlines the crucial role of the actin cortex in maintaining hindered diffusion modes of most but not all membrane molecules.

scholarly journals Nanodomains can persist at physiologic temperature in plasma membrane vesicles and be modulated by altering cell lipids

2020 ◽  
Vol 61 (5) ◽  
pp. 758-766 ◽  
Author(s):  
Guangtao Li ◽  
Qing Wang ◽  
Shinako Kakuda ◽  
Erwin London
Keyword(s):  
Plasma Membrane ◽  
Phase Separation ◽  
Light Microscopy ◽  
Large Scale ◽  
Membrane Vesicles ◽  
Lipid Domains ◽  
Domain Formation ◽  
Plasma Membrane Vesicles ◽  
Intact Cells ◽  
Wide Range

The formation and properties of liquid-ordered (Lo) lipid domains (rafts) in the plasma membrane are still poorly understood. This limits our ability to manipulate ordered lipid domain-dependent biological functions. Giant plasma membrane vesicles (GPMVs) undergo large-scale phase separations into coexisting Lo and liquid-disordered lipid domains. However, large-scale phase separation in GPMVs detected by light microscopy is observed only at low temperatures. Comparing Förster resonance energy transfer-detected versus light microscopy-detected domain formation, we found that nanodomains, domains of nanometer size, persist at temperatures up to 20°C higher than large-scale phases, up to physiologic temperature. The persistence of nanodomains at higher temperatures is consistent with previously reported theoretical calculations. To investigate the sensitivity of nanodomains to lipid composition, GPMVs were prepared from mammalian cells in which sterol, phospholipid, or sphingolipid composition in the plasma membrane outer leaflet had been altered by cyclodextrin-catalyzed lipid exchange. Lipid substitutions that stabilize or destabilize ordered domain formation in artificial lipid vesicles had a similar effect on the thermal stability of nanodomains and large-scale phase separation in GPMVs, with nanodomains persisting at higher temperatures than large-scale phases for a wide range of lipid compositions. This indicates that it is likely that plasma membrane nanodomains can form under physiologic conditions more readily than large-scale phase separation. We also conclude that membrane lipid substitutions carried out in intact cells are able to modulate the propensity of plasma membranes to form ordered domains. This implies lipid substitutions can be used to alter biological processes dependent upon ordered domains.

Sign in / Sign up

Export Citation Format

Share Document