Extraction from erythrocyte membrane

Phospholipids were extracted from erythrocyte membranes and were analyzed along with their lipoxygenase peroxide products using a 40°C aminopropyl column. Phospholipids (phosphatidylcholine [PC], phosphatidylethanolamine [PE], phospha-tidylinositol, phosphatidylserine [PS], and sphingomyelin) were separated using a 67/22/11 acetonitrile/methanol/water (0.2% TFA) mobile phase (A = 2I0nm). Elution was complete in 30 min and excellent resolution was achieved. The peroxide forms of PC, PE, and PS were separated at the same time but monitored at k — 234 nm. All samples were stabilized with 20mg/L BHT solutions [1185]. 

Historically, S-glycosylation of Cysteine was reported in two extracted peptides from human sources. A S-glycosylated octapeptide was identified from urine,and a closely-related decapeptide from erythrocytes membranes was characterized to be glycosylated on a Cysteine by a triglucoside. " However, for 40 years, these reports became controversial, as no other S-glycosylated peptides were identified.In 2011, two independent studies reported the discovery of S-glycosylated peptides (Fig. 2). 

Different tissues have different lipid compositions. The most common lipid components of membranes are PC and PE. Lipid extracts from brain and lung are also rich in PS heart tissue is rich in PG, and liver is rich in PI [567]. Human blood cells, as ghost erythrocytes (with cytoplasm contents removed), are often used as membrane models. These have different compositions between the inner and outer leaflets of the bilayer membrane. Phospholipids account for 46% of the outer leaflet membrane constituents, with PC and Sph about equal in amount. The inner leaflet is richer in phospholipids (55%), with the mix 19% PE, 12% PS, 7% PC and 5% Sph [567],... 

In 1925, E. Gorter and F. Grendel (J. Exp. Med. 41, 439) reported measurements in which they extracted lipid from red blood cell membranes with acetone, spread the lipids as a monolayer, and measured the area of the compressed monolayer. They then estimated the surface area of an erythrocyte and calculated that the ratio of the lipids (as a monolayer) to the surface area of the red blood cell was 1.9-2.0. More modern experiments gave the following each erythrocyte membrane contains 4.5 x 10 16 mol of phospholipid and 3.1 x 10-16 mol of cholesterol. 

Japanese scientists believe that the anti-oketsu effects of rhubarb are brought about by the inhibition of the production of nitric oxide, inhibition of platelet aggregation, antiallergic effects, and its antiinflammatory properties. Matsuda et al. (2001) studied and reported that the methanolic extracts from five kinds of rhubarb were found to show scavenging activity for l,l-diphenyl-2-picrylhy-drazyl (DPPH) radical and superoxide anion radical (02 ) generated by the xanthine/xanthine oxidase system and/or on lipid peroxidation by terf-butyl hydroperoxide (f-BuOOH) in the erythrocyte membrane ghost system. 

Earlier experiments have shown that cholate extracts from transformed lung fibroblasts having only the Mr 52000 subunit of Gs are active in reconstituting hormone-, nucleotide- and fluoride-stimulated adenylyl cyclase activities in cyc membranes [178]. Human erythrocyte Gs, which has only the A/r 42000 a subunit(s) [22,179], also reconstitute(s) these functions. After partial separation, rabbit liver p52 Gs appeared to reconstitute hormone-stimulated activity better than p45 Gs... 

When the lipids have been dried completely, weigh the vial again and determine the mass of lipid that you have extracted from the erythrocyte membrane. Record the mass of lipids in your notebook. 

Rabilloud, X, Blisnick, T., Heller, M., Luche, S., Aebersold, R., Limardi, J. and Braun-Breton, C. (1999) Analysis of membrane proteins by two-dimensional electrophoresis comparison of the proteins extracted from normal or Plasmodium falciparam-infected erythrocyte ghosts. Electrophoresis 20, 3603-3610. 

Proteasome research began in the late 1960s [19] when Harris discovered a barrel-shaped particle in erythrocyte extracts. From then on knowledge about the proteasome and the regulating factors of this protease has been added to step by step. Today it is generally accepted that the ubiquitin-proteasome system is responsible for the turnover of the bulk of intracellular cytosolic and nuclear proteins [20]. It is known that this protease is located in the cytosol, the nucleus, and that it is attached to the ER and other cell membranes. 

Adenylate cyclase from sperm or testis Is not stimulated by forskolin.These enzymes are not hormonally responsive and are not associated with Ns or N1 proteins. Forskolin can activate sperm membrane adenylate cyclase when extracts from human erythrocyte membranes are added to sperm membranes however. It Is not clear If the reconstitution Is revealing adenylate cyclase activity In the human erythrocyte membrane or adding back a factor required for forskolin stimulation of the sperm membrane adenylate cyclase. 5,16... 

Glycophorin can be isolated from the erythrocyte membrane by partitioning extracts produced with cither lithium diiodosalicylatc (8) or dcoxycholate (J 5) between phenol and water during which... 

Another interesting finding is that various antigens of blood type determinant accompanied by other membrane proteins were also transferred from human erythrocyte to liposome. They were respectively assayed by the turbidity increase upon the aggregation of liposome with the specific antibody. All the modified liposomes, except simple DMPC liposome, extracted A-type antigen from erythrocyte (Table 2). The sensitivity of assay of the antigen on the liposome, which... 

Figure 9.12 Direct electrospray ionization-mass spectrometry analysis of human erythrocyte plasma membrane phospholipids (A) A positive-ion electrospray ionization (ESI) mass spectrum of erythrocyte plasma membrane phospholipid extract showing 14 molecular species of glycerophospholipids and 4 molecular species of sphingomyelin (B) A negative-ion ESI mass spectrum of the same extract of plasma membrane phospholipids showing more than 25 molecular species of ethanolamine glycerophospholipids and 8 molecular species of serine and inositol glycerophospholipids. Reprinted with permission from Han, X. and Gross, R. W., Electrospray ionization mass spectroscopic analysis of human erythrocyte plasma membrane phospholipids, Proc. Natl Acad. Scl USA, 91(22), 10635-9. Copyright (1994) National Academy of Sciences, USA.

Interestingly, plasma membrane NADH-AFR reductase is inhibited by detergents at concentrations that promote membrane solubilization and cannot then be recovered from the extract, even after removal of detergent. This inhibition is not likely to be due to delipidation of a single transmembrane protein, since addition of sonicated purified phospholipids to the reaction mixture does not restore the activity. These results support the interpretation that the electron flow from NADH to AFR appears to involve more than one protein carrier (Villalba et al., 1993a). NADH-ferricyanide reductase has been purified from erythrocyte plasma membranes as a single polypeptide of 36 kDa and shown to correspond to NADH-cytochrome reductase (Kitajima et al., 1981). Its activity is not affected by WGA (Villalba a/., 1993a). 

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