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 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.

Evidence for a selective and electroneutral K+/H+-exchange in Saccharomyces cerevisiae using plasma membrane vesicles

Yeast ◽  
1996 ◽  
Vol 12 (13) ◽  
pp. 1301-1313 ◽  
Author(s):  
Carole Camarasa ◽  
Susana Prieto ◽  
Roc Ros ◽  
Jean-Michel Salmon ◽  
Pierre Barre
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles

scholarly journals Study of fluxes at low concentrations of l-tri-iodothyronine with rat liver cells and their plasma-membrane vesicles. Evidence for the accumulation of the hormone against a gradient

1981 ◽  
Vol 198 (3) ◽  
pp. 457-466 ◽  
Author(s):  
Govind S. Rao ◽  
Marie Luise Rao ◽  
Astrid Thilmann ◽  
Hans D. Quednau
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Liver Homogenate ◽  
Membrane Vesicles ◽  
Liver Cells ◽  
Free Form ◽  
Plasma Membrane Vesicles ◽  
Cytosol Fraction ◽  
Low Concentrations ◽  
Rat Liver Cells

1. Influx and efflux of l-tri-[125I]iodothyronine with isolated rat liver parenchymal cells and their plasma-membrane vesicles were studied by a rapid centrifugation technique. 2. At 23°C and in the concentration range that included the concentration of free l-tri-iodothyronine in rat plasma (3–5pm) influx into cells was saturable; an apparent Kt value of 8.6±1.6pm was obtained. 3. At 5pm-l-tri-[125I]iodothyronine in the external medium the ratios of the concentrations inside to outside in cells and plasma-membrane vesicles were 38:1 and 366:1 respectively after 7s of incubation. At equilibrium (60s at 23°C) uptake of l-tri-[125I]iodothyronine by cells was linear with the hormone concentration, whereas that by plasma-membrane vesicles exhibited an apparent saturation with a Kd value of 6.1±1.3pm. 4. Efflux of l-tri-[125I]iodothyronine from cells equilibrated with the hormone (5–123pm) was constant up to 21 s; the amount that flowed out was 17.7±3.8% when cells were equilibrated with 5pm-hormone. When plasma-membrane vesicles were equilibrated with l-tri-[125I]iodothyronine (556–1226pm) 66.8±5.8% flowed out after 21 s. 5. From a consideration of the data on efflux from cells and binding of l-tri-[125I]iodothyronine to the liver homogenate, as studied by the charcoal-adsorption and equilibrium-dialysis methods, it appears that 18–22% of the hormone exists in the free form in the cell. 6. Vinblastine and colchicine diminished the uptake of l-tri-[125I]iodothyronine by cells but not by plasma-membrane vesicles; binding to the cytosol fraction was not affected. Phenylbutazone, 6-n-propyl-2-thiouracil, methimazole and corticosterone diminished the uptake by cells, plasma-membrane vesicles and binding to the cytosol fraction to different extents. 7. These results suggest that at low concentrations of l-tri-[125I]iodothyronine rat liver cells and their plasma-membrane vesicles accumulated the hormone against an apparent gradient by a membrane-mediated process. Contribution of cytoplasmic proteins to uptake by plasma-membrane vesicles was negligible. The amount of l-tri-[125I]iodothyronine required to achieve half-maximal uptake agrees with that occurring in the free form in the blood, conferring physiological importance to the transporting system in the plasma membrane of the liver cell.

Structural studies of purified pig lymph node plasma membrane: association of cytoskeletal components with the plasma membrane and the effect of detergent solubilization

1982 ◽  
Vol 60 (1) ◽  
pp. 57-70 ◽  
Author(s):  
Robert J. Allore ◽  
Brian H. Barber
Keyword(s):  
Plasma Membrane ◽  
Lymph Node ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Membrane Vesicles ◽  
Sodium Deoxycholate ◽  
Insoluble Fraction ◽  
Plasma Membrane Vesicles ◽  
Detergent Solubilization ◽  
Lentil Lectin

The reproducibility of preparation, stability at 4 °C, and detergent solubilization characteristics of plasma membrane vesicles purified from domestic pig mesenteric lymph node tissue have been examined. It was found mat 2% (w/v) Nonidet P-40 solubilized 50–60% and 2% (w/v) sodium deoxycholate solubilized 60–70% of the total membrane protein. As judged by 125I-labelled lentil lectin staining of the sodium dodecyl sulfate – polyacrylamide gel electrophoresis patterns, 2% (w/v) Nonidet P-40 solubilized approximately 73%, and 2% (w/v) sodium deoxycholate approximately 82% of the total glycoprotein. Actin and a myosin-like component were identified as major constituents of both the Nonidet P-40 and the sodium deoxycholate insoluble fractions, suggesting the possibility that the detergent-insoluble fraction may represent a membrane-associated cytoskeletal network analogous to that which has been demonstrated for the erythrocyte membrane. Consistent with such an intimate association between actin and the plasma membrane, it was found mat very little actin could be eluted from the intact membrane vesicles by dialysis against low ionic strength ATP solutions, 0.6 M KCl, or by incubation with DNase I.

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