scholarly journals Purification of the NF2 Tumor Suppressor Protein from Human Erythrocytes

2006 ◽  
Vol 33 (4) ◽  
pp. 394-402 ◽  
Author(s):  
Hitesh K. Jindal ◽  
Kazumi Yoshinaga ◽  
Pil-Soo Seo ◽  
Mohini Lutchman ◽  
Patrick A. Dion ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Tumor Suppressor ◽  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Mammalian Cells ◽  
Tumor Suppressor Protein ◽  
Human Erythrocyte Membrane ◽  
Protein 4.1 ◽  
Erythrocyte Plasma Membrane ◽  
Inside Out

Background:Neurofibromatosis type 2 (NF2) is an autosomal dominant disease predisposing individuals to the risk of developing tumors of cranial and spinal nerves. The NF2 tumor suppressor protein, known as Merlin/Schwanomin, is a member of the protein 4.1 superfamily that function as links between the cytoskeleton and the plasma membrane.Methods:Upon selective extraction of membrane-associated proteins from erythrocyte plasma membrane (ghosts) using low ionic strength solution, the bulk of NF2 protein remains associated with the spectrin-actin depleted inside-out-vesicles. Western blot analysis showed a ~70 kDa polypeptide in the erythrocyte plasma membrane. Furthermore, quantitative removal of NF2 protein from the inside-out-vesicles was achieved using 1.0 M potassium iodide, a treatment known to remove tightly-bound peripheral membrane proteins.Results:These results suggest a novel mode of NF2 protein association with the erythrocyte membrane that is distinct from the known membrane interactions of protein 4.1. Based on these biochemical properties, several purification strategies were devised to isolate native NF2 protein from human erythrocyte ghosts. Using purified and recombinant NF2 protein as internal standards, we quantified approximately ~41-65,000 molecules of NF2 protein per erythrocyte.Conclusion:We provide evidence for the presence of NF2 protein in the human erythrocyte membrane. The identification of NF2 protein in the human erythrocyte membrane will make it feasible to discover novel interactions of NF2 protein utilizing powerful techniques of erythrocyte biochemistry and genetics in mammalian cells.

scholarly journals Changes in Human Erythrocyte Membrane Exposed to Aqueous and Ethanolic Extracts from Uncaria tomentosa

Molecules ◽  
2021 ◽  
Vol 26 (11) ◽  
pp. 3189
Author(s):  
Piotr Duchnowicz ◽  
Radosław Pilarski ◽  
Jaromir Michałowicz ◽  
Bożena Bukowska
Keyword(s):  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Ethanolic Extract ◽  
Blood Purification ◽  
Human Erythrocyte Membrane ◽  
Uncaria Tomentosa ◽  
Erythrocyte Shape ◽  
Ethanolic Extracts ◽  
Erythrocyte Membrane Fluidity ◽  
Erythrocyte Plasma Membrane

Uncaria tomentosa (Willd.) DC is a woody climber species originating from South and Central America that has been used in the therapy of asthma, rheumatism, hypertension, and blood purification. Our previous study showed that U. tomentosa extracts altered human erythrocyte shape, which could be due to incorporation of the compounds contained in extracts into the erythrocyte membrane. The aim of the present study was to determine how the compounds contained in U. tomentosa extracts incorporate into the human erythrocyte membrane. The study has assessed the effect of aqueous and ethanolic extracts from leaves and bark of U. tomentosa on the osmotic resistance of the human erythrocyte, the viscosity of erythrocyte interior, and the fluidity of erythrocyte plasma membrane. Human erythrocytes were incubated with the studied extracts in the concentrations of 100, 250, and 500 µg/mL for 2, 5, and 24 h. All extracts tested caused a decrease in erythrocyte membrane fluidity and increased erythrocyte osmotic sensitivity. The ethanolic extracts from the bark and leaves increased viscosity of the erythrocytes. The largest changes in the studied parameters were observed in the cells incubated with bark ethanolic extract. We consider that the compounds from U. tomentosa extracts mainly build into the outer, hydrophilic monolayer of the erythrocyte membrane, thus protecting the erythrocytes against the adverse effects of oxidative stress.

scholarly journals Electron microscope study of reassociation of spectrin and actin with the human erythrocyte membrane

1981 ◽  
Vol 90 (1) ◽  
pp. 70-77 ◽  
Author(s):  
S Tsukita ◽  
S Tsukita ◽  
H Ishikawa ◽  
S Sato ◽  
M Nakao
Keyword(s):  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Erythrocyte Membranes ◽  
Human Erythrocyte Membrane ◽  
Thin Sections ◽  
Fixation Treatment ◽  
Human Erythrocyte Membranes ◽  
Inside Out

Reassociation of spectrin and actin with human erythrocyte membranes was studied by stereoscopic electron microscopy of thin sections combined with tannic acid- glutaraldehyde fixation. Treatment of the erythrocyte membrane with 0.1 mM EDTA (pH 8.0) extracted more than 90 percent of the spectrin and actin and concomitantly removed filamentous meshworks underlying the membranes, followed by fragmentation into small inside-out vesicles. When such spectrin-depleted vesicles were incubated with the EDTA extract (crude spectrin), a filamentous meshwork, similar to those of the original membranes, was reformed on the cytoplasmic surface of the vesicles. The filamentous components, with a uniform thickness of 9 nm, took a tortuous course and joined one another often in an end-to-end fashion to form a irregular but continuous meshwork parallel to the membrane. Purified spectrin was also reassociated with the vesicles in a population density of filamentous components almost comparable to that of the crude spectrin-reassociated vesicles. However, the meshwork formation was much smaller in extent, showing many independent filamentous components closely applied to the vesicle surface. When muscle G-actin was added to the crude spectrin- or purified spectrin- reassociated vesicles under conditions which favor actin polymerization, actin filaments were seen to attach to the vesicles through the filamentous components. Two modes of association of actin filaments with the membrane were seen: end-to-membrane and side-to- membrane associations. In the end-to-membrane association, each actin filament was bound with several filamentous components exhibiting a spiderlike configuration, which was considered to be the unit of the filamentous meshwork of the original erythrocyte membrane.

scholarly journals Analysis of Receptor for Vibrio choleraeEl Tor Hemolysin with a Monoclonal Antibody That Recognizes Glycophorin B of Human Erythrocyte Membrane

1999 ◽  
Vol 67 (10) ◽  
pp. 5332-5337 ◽  
Author(s):  
Dongyan Zhang ◽  
Junko Takahashi ◽  
Taiko Seno ◽  
Yoshihiko Tani ◽  
Takeshi Honda
Keyword(s):  
Monoclonal Antibody ◽  
Vibrio Cholerae ◽  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Mammalian Cells ◽  
Biochemical Characterization ◽  
Human Erythrocytes ◽  
Human Erythrocyte Membrane ◽  
El Tor ◽  
Glycophorin B

ABSTRACT El Tor hemolysin (ETH), a pore-forming toxin secreted byVibrio cholerae O1 biotype El Tor and most Vibrio cholerae non-O1 isolates, is able to lyse erythrocytes and other mammalian cells. To study the receptor for this toxin or the related molecule(s) on erythrocyte, we first isolated a monoclonal antibody, B1, against human erythrocyte membrane, which not only blocks the binding of ETH to human erythrocyte but also inhibits the hemolytic activity of ETH. Biochemical characterization and immunoblotting revealed that this antibody recognized an epitope on the extracellular domain of glycophorin B, a sialoglycoprotein of erythrocyte membrane. Erythrocytes lacking glycophorin B but not glycophorin A were less sensitive to the toxin than were normal human erythrocytes. These results indicate that glycophorin B is a receptor for ETH or at least an associated molecule of the receptor for ETH on human erythrocytes.

Evidence for an Asymmetric Distribution of Phospholipids in the Human Erythrocyte Membrane

1974 ◽  
Vol 52 (9) ◽  
pp. 803-806 ◽  
Author(s):  
Arthur Kahlenberg ◽  
Caroline Walker ◽  
Ruth Rohrlick
Keyword(s):  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Phospholipid Composition ◽  
Enzyme Hydrolysis ◽  
Asymmetric Distribution ◽  
Human Erythrocyte Membrane ◽  
Cytoplasmic Surface ◽  
Direct Demonstration ◽  
Inside Out ◽  
Phospholipases A

The changes in phospholipid composition of the inner (cytoplasmic) surface of the human erythrocyte membrane resulting from the digestion of sealed inside-out vesicles with phospholipases A2 and C were determined. Virtually all of the phosphatidylethanolamine and phosphatidylserine and 30–40% of the phosphatidylcholine and sphingomyelin of inside-out vesicles were found to be accessible to enzyme hydrolysis. In contrast, all of the above phospholipids of unsealed ghosts were susceptible to phospholipolytic digestion. These results are a direct demonstration of an asymmetric distribution of phospholipids in the human erythrocyte membrane.

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