scholarly journals Purification of the Plasma Membrane Ca2+-ATPase from Radish Seedlings by Calmodulin-Agarose Affinity Chromatography

1998 ◽  
Vol 116 (2) ◽  
pp. 845-851 ◽  
Author(s):  
Cristina Bonza ◽  
Antonella Carnelli ◽  
Maria Ida De Michelis ◽  
Franca Rasi-Caldogno
Keyword(s):  
Plasma Membrane ◽  
Affinity Chromatography ◽  
Radish Seedlings

Isolation Of Plasma Membrane Vesicles Of Human Platelet By Affinity Chromatography

1981 ◽  
Author(s):  
T Kobayashi ◽  
M Sakon ◽  
H Ohno ◽  
J Kambayshi ◽  
G Kösaki
Keyword(s):  
Plasma Membrane ◽  
Affinity Chromatography ◽  
Morphological Changes ◽  
Membrane Vesicles ◽  
Dry Weight ◽  
Appreciable Amount ◽  
Marker Enzyme ◽  
Plasma Membrane Vesicles ◽  
Platelet Suspension ◽  
Centrifugal Method

Platelets undergo a unique morphological changes leading to the formation of hemostatic plug. In recent years, its intermediaty metabolism has been extensively studied and the important function of plasma membrane in the platelet reaction has been recognized. The method of Barber and Jamieson has been employed in order to prepare plasma membrane vesicles of platelet of excellent quality but it is rather time consuming and the yield is relatively low. In this study, an attempt was made to isolate plasma membrane vesicles of human platelets by wheat germ agglutinin affinity chromatography.Freshly collected human citrated blood was subjected to glycerol loading and hypotonic lysis to obtain lysed platelet suspension. Then, it was applied to the affinity chromatography and the fraction of plasma membrane vesicles was eluted by 0.2 M N-acetyl glucosamine. Electron micrograph of the fraction showed round membrane vesicles with some scattered intracellular organelles. Several marker enzymes were assayed in the fraction. No appreciable amount of β-glucuronidase or cytochrome c oxidase was detected in the fraction, indicating no contamination of mitochondria or α-granules. Relatively high activity of G-6-Pase was detected, suggesting possible contamination of endoplasmic reticulum. The yield was 11.6% in dry weight and 7.9% in protein.By this method, the isolation was much faster than the centrifugal method and as low as 20 ml of human citrated whole blood may be used as starting material. Upon characterization of the plasma membrane fraction by electron microscopy and marker enzyme assays, the quality of the fraction was found comparable with the centrifugal method. The yield by this method was approximately two times higher than by the conventional method.

scholarly journals Isolation of the full-length murine erythropoietin receptor using a baculovirus expression system

Blood ◽  
1996 ◽  
Vol 87 (3) ◽  
pp. 926-937 ◽  
Author(s):  
JL Spivak ◽  
LS Avedissian ◽  
JH Pierce ◽  
D Williams ◽  
WD Hankins ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Affinity Chromatography ◽  
Molecular Mass ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Erythropoietin Receptor ◽  
Receptor Protein ◽  
Sf9 Cells ◽  
Con A ◽  
Whole Cell

The full-length murine erythropoietin receptor was expressed in Spodoptera frugiperda (Sf9) cells using a recombinant baculovirus vector. Erythropoietin receptor protein production was maximal 48 hours after infection, as determined by metabolic labeling and immunoblotting; receptor protein varied in molecular mass from 62 to 76 kD. Erythropoietin receptors produced in Sf9 cells could be solubilized using CHAPS in a form capable of binding erythropoietin, and the solubilized receptor bound to immobilized Concanavalin A (Con A) and wheat germ agglutinin, as well as to immobilized recombinant human erythropoietin. Analysis of the distribution of erythropoietin receptors in Sf9 plasma membrane and cytosol fractions using lectin affinity chromatography revealed that membrane-bound receptor had a higher apparent molecular mass and contained the bulk of receptors that bound to wheat germ agglutinin. The receptor was purified by sequential affinity chromatography on Con A-Sepharose and immobilized erythropoietin. Erythropoietin receptors expressed in Sf9 cells were inserted into the plasma membrane in the correct orientation, bound 125I-erythropoietin with a single affinity (kD, 330 pmol/L), and were internalized after ligand binding. However, kD varied inversely with the number of cell surface receptors. Solubilized erythropoietin receptors in whole-cell lysates and isolated plasma membranes exhibited high-affinity binding, with kD values of 92 and 57 pmol/L, respectively. Erythropoietin bound to the surface of infected Sf9 cells could be cross-linked to two proteins with molecular masses of 90 and 65 kD using the homobifunctional cross-linker, disuccinimidyl suberate (DSS). Similar results were obtained with solubilized receptors in whole-cell lysates, and both proteins could be immunoprecipitated by an antiserum to the erythropoietin receptor carboxyl-terminal domain.

scholarly journals Plasma Membrane Ca-ATPase of Radish Seedlings

1992 ◽  
Vol 98 (3) ◽  
pp. 1196-1201 ◽  
Author(s):  
Antonella Carnelli ◽  
Maria I. De Michelis ◽  
Franca Rasi-Caldogno
Keyword(s):  
Plasma Membrane ◽  
Radish Seedlings

Isolation of plasma membrane vesicles of human platelets by affinity chromatography

1981 ◽  
Vol 21 (1-2) ◽  
pp. 129-135 ◽  
Author(s):  
J. Kambayashi ◽  
M. Sakon ◽  
H. Ohno ◽  
G. Kòsaki
Keyword(s):  
Plasma Membrane ◽  
Affinity Chromatography ◽  
Membrane Vesicles ◽  
Human Platelets ◽  
Plasma Membrane Vesicles

Multiple Effects of Lysophosphatidylcholine on the Activity of the Plasma Membrane H+-ATPase of Radish Seedlings*

Botanica Acta ◽  
1997 ◽  
Vol 110 (1) ◽  
pp. 43-48 ◽  
Author(s):  
Maria Ida De Michelis ◽  
R. Papini ◽  
Maria Chiara Pugliarello
Keyword(s):  
Plasma Membrane ◽  
Radish Seedlings
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