scholarly journals High-yield isolation of functionally competent endosomes from mouse lymphocytes

1989 ◽  
Vol 264 (1) ◽  
pp. 137-149 ◽  
Author(s):  
B D Beaumelle ◽  
C R Hopkins
Keyword(s):  
Plasma Membrane ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Membrane Vesicles ◽  
High Yield ◽  
Plasma Membrane Vesicles ◽  
Free System ◽  
Protein Factor ◽  
Early Endosomes ◽  
Discontinuous Sucrose Gradient

A discontinuous-sucrose-gradient procedure for isolating endosomes from mouse lymphoma cells has been developed. After centrifugation, most organelles (especially mitochondria and lysosomes) are recovered in the denser fractions of the gradient, whereas a mixture of plasma membrane and endosomes is present at lighter densities. The endosome recovery in this fraction can be increased (by 100%) by (a) a mild trypsin treatment of the postnuclear supernatant and (b) loading the cell endosomes with a saturating concentration of low-density lipoproteins. Removal of the plasma-membrane contamination was achieved by preincubating the cells with a gold-ricin complex at 4 degrees C. On centrifugation, the gold-loaded membranes sediment to the bottom of the gradient. The endosome preparation isolated by these procedures is less than 6% contaminated by other organelles and contains 42% of internalized 125I-transferrin. We show that these isolated endosomes are functional, as displayed by their ability to fuse and to acidify in a cell-free system. Endosome fusion was studied by a new assay based on the use of fluorescence resonance energy transfer. This fusion is dependent on ATP and on a cytosolic, thermoresistant but trypsin- and N-ethylmaleimide-sensitive, protein factor. Early endosomes fuse more actively among themselves than with late-endocytic vesicles, and they fuse only slowly with plasma-membrane vesicles.

scholarly journals Neutrophil plasma membranes. I. High-yield purification of human neutrophil plasma membrane vesicles by nitrogen cavitation and differential centrifugation.

1980 ◽  
Vol 86 (1) ◽  
pp. 21-28 ◽  
Author(s):  
M S Klempner ◽  
R B Mikkelsen ◽  
D H Corfman ◽  
J André-Schwartz
Keyword(s):  
Plasma Membrane ◽  
Human Neutrophil ◽  
Plasma Membranes ◽  
Hydrolytic Enzymes ◽  
Membrane Vesicles ◽  
Galactose Oxidase ◽  
High Yield ◽  
Differential Centrifugation ◽  
Plasma Membrane Vesicles ◽  
Equilibrium Ultracentrifugation

Neutrophil chemotaxis, phagocytosis, and oxygen-dependent microbicidal activity are initiated by interactions of stimuli with the plasma membrane. However, difficulties in neutrophil plasma membrane isolation have precluded studies on the precise structure or function of this cellular component. In this paper, a method is described for the isolation of representative human neutrophil plasma membrane vesicles, using nitrogen cavitation for cell disruption and a combination of differential centrifugation and equilibrium ultracentrifugation in Dextran gradients for membrane fractionation. Multiple biochemical markers and galactose oxidase-tritiated sodium borohydride surface labeling were employed to follow the yield, purity, and distribution of plasma membranes, nuclei, lysosomes, endoplasmic reticulum, mitochondria, and cytosol. According to these markers, neutrophil plasma membranes were exposed to minimal lysosomal hydrolytic enzymes and could be isolated free of other subcellular organelles. In contrast, disruption of neutrophils by mechanical homogenization resulted in > 20% lysosomal rupture and significant plasma membrane proteolysis. Electron microscopy demonstrated that plasma membranes isolated after nitrogen cavitation appeared to be sealed vesicles with striking homogeneity.

scholarly journals Structural and functional polarity of canalicular and basolateral plasma membrane vesicles isolated in high yield from rat liver.

1984 ◽  
Vol 98 (3) ◽  
pp. 991-1000 ◽  
Author(s):  
P J Meier ◽  
E S Sztul ◽  
A Reuben ◽  
J L Boyer
Keyword(s):  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Rat Liver ◽  
High Speed ◽  
Membrane Vesicles ◽  
Fold Increase ◽  
High Yield ◽  
Marker Enzyme ◽  
Gamma Glutamyl Transpeptidase ◽  
Plasma Membrane Vesicles

A method has been developed for routine high yield separation of canalicular (cLPM) from basolateral (blLPM) liver plasma membrane vesicles of rat liver. Using a combination of rate zonal floatation (TZ-28 zonal rotor, Sorvall) and high speed centrifugation through discontinuous sucrose gradients, 9-16 mg of cLPM and 15-28 mg of blLPM protein can be isolated in 1 d. cLPM are free of the basolateral markers Na+/K+-ATPase and glucagon-stimulatable adenylate cyclase activities, but are highly enriched with respect to homogenate in the "canalicular marker" enzyme activities leucylnaphthylamidase (48-fold), gamma-glutamyl-transpeptidase (60-fold), 5'-nucleotidase (64-fold), alkaline phosphatase (71-fold), Mg++-ATPase (83-fold), and alkaline phosphodiesterase I (116-fold). In contrast, blLPM are 34-fold enriched in Na+/K+-ATPase activity, exhibit considerable glucagon-stimulatable adenylate cyclase activity, and demonstrate a 4- to 15-fold increase over homogenate in the various "canalicular markers." cLPM have a twofold higher content of sialic acids, cholesterol; and sphingomyelin compared with blLPM. At least three canalicular-(130,000, 100,000, and 58,000 mol wt) and several basolateral-specific protein bands have been detected after SDS PAGE of the two LPM subfractions. Specifically, the immunoglobin A-binding secretory component is restricted to blLPM as demonstrated by immunochemical techniques. These data indicate virtually complete separation of basolateral from canalicular LPM and demonstrate multiple functional and compositional polarity between the two surface domains of hepatocytes.

scholarly journals BRET-based effector membrane translocation assay monitors GPCR-promoted and endocytosis-mediated Gq activation at early endosomes

2021 ◽  
Vol 118 (20) ◽  
pp. e2025846118
Author(s):  
Shane C. Wright ◽  
Viktoriya Lukasheva ◽  
Christian Le Gouill ◽  
Hiroyuki Kobayashi ◽  
Billy Breton ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Protein Translocation ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Signal Propagation ◽  
Membrane Translocation ◽  
Receptor Endocytosis ◽  
Early Endosomes ◽  
Translocation Assay

G protein–coupled receptors (GPCRs) are gatekeepers of cellular homeostasis and the targets of a large proportion of drugs. In addition to their signaling activity at the plasma membrane, it has been proposed that their actions may result from translocation and activation of G proteins at endomembranes—namely endosomes. This could have a significant impact on our understanding of how signals from GPCR-targeting drugs are propagated within the cell. However, little is known about the mechanisms that drive G protein movement and activation in subcellular compartments. Using bioluminescence resonance energy transfer (BRET)–based effector membrane translocation assays, we dissected the mechanisms underlying endosomal Gq trafficking and activity following activation of Gq-coupled receptors, including the angiotensin II type 1, bradykinin B2, oxytocin, thromboxane A2 alpha isoform, and muscarinic acetylcholine M3 receptors. Our data reveal that GPCR-promoted activation of Gq at the plasma membrane induces its translocation to endosomes independently of β-arrestin engagement and receptor endocytosis. In contrast, Gq activity at endosomes was found to rely on both receptor endocytosis-dependent and -independent mechanisms. In addition to shedding light on the molecular processes controlling subcellular Gq signaling, our study provides a set of tools that will be generally applicable to the study of G protein translocation and activation at endosomes and other subcellular organelles, as well as the contribution of signal propagation to drug action.

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 Sodium-gradient-stimulated transport of l-alanine by plasma-membrane vesicles isolated from liver parenchymal cells of fed and starved rats. Crucial role of the adrenal glucocorticoids

1982 ◽  
Vol 208 (3) ◽  
pp. 685-693 ◽  
Author(s):  
Dennis C. Quinlan ◽  
C. Gordon Todderud ◽  
Darshan S. Kelley ◽  
Rolf F. Kletzien
Keyword(s):  
Plasma Membrane ◽  
Active Transport ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicle ◽  
Plasma Membrane Vesicles ◽  
Free System ◽  
Liver Parenchymal ◽  
Alanine Transport ◽  
Parenchymal Cells

The ability of liver efficiently to take up amino acids, particularly l-alanine, during starvation was studied in a cell-free system by isolating plasma-membrane vesicles in a transport-competent state from rat liver parenchymal cells. These membrane vesicles have the capacity to accumulate l-alanine against an apparent concentration gradient when exposed to an artificial and transient transmembrane Na+ gradient (extravesicular Na+ concentration greater than inside). The rate of accumulation of l-alanine is dependent on the plasma-membrane vesicle concentration, and the steady-state concentration attained is inversely related to the osmolarity of the medium. The Na+-mediated stimulation is not exhibited if the membrane vesicles are pre-equilibrated with NaCl, if K+ or Li+ are substituted for Na+, or if SO42− replaces Cl− as the counterion. The apparent active transport of l-alanine into the membrane vesicles appears to occur by an electrogenic mechanism: (1) the use of NaSCN significantly heightens the early concentrative phase of transport when compared with the effect of NaCl; (2) an enhanced active transport is also observed when a valinomycin-induced K+ efflux occurs concomitant with Na+ and l-alanine influx. Plasma-membrane vesicles isolated from liver parenchymal cells of a 24 h-starved rat exhibit an initial l-alanine transport rate that is 3–4 times that for membrane vesicles derived from a fed animal. The increased rate of l-alanine transport by plasma-membrane vesicles from starved animals can be obliterated by adrenalectomy and restored by administration of glucocorticoid. These results establish that stimulation of the gluconeogenic pathway by starvation involves a plasma-membrane-localized change affecting l-alanine transport which is regulated in part by the glucocorticoid hormones.

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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