Characterization of the oligosaccharide units of the bovine erythrocyte membrane glycoprotein.

The major glycoprotein of the bovine erythrocyte membrane was purified by extraction of the ghosts with lithium 3,5-diiodosalicylate followed by phenol-water extraction and acidification. The glycoprotein contains 20% protein and 80% carbohydrate by weight and gives a single band on sodium dodecyl sulfate-polyacrylamide gels with an estimated molecular weight of 230000 daltons. The carbohydrate composition of the glycoprotein was determined to be (in residues relative to sialic acid): sialic acid, 1.0; fucose, less than 0.01; mannose, 0.1; galactose, 3.3; N-acetylgalactosamine, 0.9; and N-acetylglucosamine, 2.4. Pronase digestion of the isolated glycoprotein followed by Sephadex G-75 gel filtration resulted in the separation of a small pool of glycopeptides (pool III), which included all of the mannose-containing glycopeptides, from the bulk of the glycopeptide material which was in the void fractions of the column (pool I). Alkaline borohydride treatment released over 95% of the oligosaccharide units in pool I and approximately 30% of the oligosaccharide units in pool III. These oligosaccharides were isolated by gel filtration and ion-exchange chromatography. The oligosaccharides released from pool I had molecular weights of 1100-1400 daltons and contained sialic acid, galactose, and N-acetylglucosamine in molar ratios of 0.5-1:3:2 as well as a partial residue of N-acetylgalactosaminitol. The oligosaccharides released from pool III by alkali had molecular weights of 1300-1600 daltons and contained sialic acid, galactose, N-acetylglucosamine, N-acetylgalactosamine and N-ACETYLgalactosaminitol in molar ratios of 1-2:2:1:1:1. These data indicate that the majority of the oligosaccharide units of the bovine erythrocyte glycoprotein are linked O-glycosidically to the peptide backbone of the molecule.     

Oscillations in bovine erythrocyte membrane free thiols

Oscillations in the level of bovine erythrocyte membrane-SH groups available for DTNB are described. The concentrations of slow- and fast-reactive thiols and the kinetic properties of slow and fast reactions between -SH and DTNB revealed two kinds of cyclic changes. Oscillations of short (about 3 h) and long (about 14 h) duration were studied and the possible implications of these variations in membranes are discussed.     

Characterization of gangliosides from bovine erythrocyte membranes

Two glucosamine-containing gangliosides, sialosylhexaglycosylceramides, were isolated from bovine erythrocyte membranes. Both gangliosides were hydrolyzed by neuraminidase isolated from Clostridium perfringens to become neutral hexaglycosylceramides. Based on the results of sequential enzymatic hydrolysis and gas chromatography-mass spectrometric analyses of the methylated sugars, the structures of these two gangliosides were shown to be NeuAcalpha2 leads to 3Galbeta1 leads to 4GlcNAcbeta1 leads to 3Galbeta1 leads to 4GlcNAcbeta1 leads to 3Galbeta1 leads to 4Glc-ceramide and NeuGcalpha2 leads to 3Galbeta1 leads to 4GlcNAcbeta1 leads to 3Galbeta1 leads to 4GlcNAcbeta1 leads to 3Galbeta1 leads to 4Glc-ceramide, respectively. In addition, N-acetyl- and N-glycolylneuraminosyllacto-N-neotetraosylceramides, and N-acetyl- and N-glycolylneuraminosyllactosylceramides were also found in bovine erythrocytes. The predominant fatty acids in these two gangliosides were C 22:0 and C 24:0. C-18 sphingosine was the major base detected.     

Characterization of human sperm plasma membrane: glycolipids and polypeptides

The plasma membranes from ejaculated human spermatozoa were removed by nitrogen cavitation (600 PSI for 10 min) and isolated by centrifugation followed by a discontinuous sucrose density gradient centrifugation. Glycolipid analysis of the plasma membrane revealed a three-fold enrichment in gangliosides: GM3 and GD1a/GD1b and neutral glycolipids: globoside and sulfatide as compared to that of whole human sperm. Two dimensional electrophoresis of human sperm plasma membranes revealed about 75 polypeptides. Several of these polypeptides were similar in migration and in display of shape and color to that found in boar sperm plasma membranes.     

The structure of the major protein of the human erythrocyte membrane. Characterization of the intact protein and major fragments.

Polypeptide 3, the major membrane-penetrating protein of the human erythrocyte membrane, was characterized, together with two major fragments derived by specific proteolysis of the native protein in the membrane. One fragment (fragment 3f) was obtained from thermolysin cleavage in the extracellular region of the protein, and the other (fragment T1) was derived from tryptic cleavage in the intracellular region of the protein. The results of N- and C-terminal group analysis suggest that fragment 3f contains the N-terminal region of polypeptide 3 and fragment T1 contains the C-terminal part of the molecule. The carbohydrate contents of the polypeptides suggest that carbohydrates are present in three regions of the molecule, much of this carbohydrate being present in the C-terminal part of the molecule. This region of the protein also contains the receptors for concanavalin and the lectins from Phaseolus vulgaris and Ricinis communis, and our results suggest that there is heterogeneity in the carbohydrate chains present in the C-terminal region of polypeptide 3. These data are related to the folding of polypeptide 3 in the erythrocyte membrane.     

Characterization of calpain I-binding proteins in human erythrocyte plasma membrane

The calpain-binding components on the plasma membrane were characterized using calpain I. 125I-labeled calpain was bound to inside-out membrane vesicles from human erythrocyte in a Ca2(+)-dependent manner, but not to right-side-out membrane vesicles. The maximum binding was observed at more than 5 microM Ca2+. The binding amount of calpain to the inside-out membrane vesicles was decreased when the vesicles were pretreated with 100 micrograms/ml of trypsin, chymotrypsin, elastase, or pronase P for 30 min at 37 degrees C, although the binding to the vesicles pretreated with 200 micrograms/ml of phospholipase A2 or C was not affected. Calpain-binding proteins in the membrane were analyzed by using a modified immunoblotting for calpain. Immunostained bands of 240, 220, 89, 72, 52, and 36 kDa were detected as the calpain-binding proteins in the native membrane. All of these bands had disappeared in trypsin-treated membrane. The disappearance of bands was dose-dependent with respect to trypsin and paralleled the reduction of binding amount of calpain to the trypsinized membrane. In calpain-treated membrane, the 240 and 36 kDa bands were retained in the blotting, though the other bands disappeared dose-dependently with respect to calpain. These results suggested that the significant component in the inner surface of plasma membrane for binding of calpain was proteinaceous and the calpain-binding proteins could be classified into two species, i.e. substrates of calpain (220, 89, 72, and 52 kDa protein) and non-substrates (240 and 36 kDa protein).     

Capillary gel electrophoresis: separation of major erythrocyte membrane proteins

A new separation method of human erythrocyte membrane proteins by sodium dodecyl sulfate capillary gel electrophoresis (SDS–CGE) is described. In this method, a replaceable gel matrix was used. Seven major erythrocyte membrane proteins, α-and β-spectrin, ankyrin 2.1, band 3 (anion-exchanger), 4.1a and b, and 4.2 (pallidin), were separated and identified by SDS–CGE method. High reproducible migration times of these proteins (inter-assay coefficients of variation less than 2%), as well as quantification (inter-assay coefficients of variation less than 11%) were obtained. This new SDS–CGE method may provide important diagnostic evidence for hereditary spherocytosis. It can be a powerful diagnostic tool in place of SDS polyacrylamide gel electrophoresis for erythrocyte membrane protein analysis.     

Purification and characterization of carbonic anhydrase from bovine erythrocyte plasma membrane

Carbonic anhydrase (CA) was purified from bovine erythrocyte plasma membrane and characterized in this study. For this purpose, the blood taken from young animals was hemolysed, the membrane fraction was separated, and this fraction was repeatedly washed. The enzyme (CA) was removed from the membrane with buffered TritonX-100 (1%); it could be purified at a factor of 22.8 by affinity chromatography.The CA obtained from erythrocyte membrane has an esterase activity as well as hydratase activity. The Vmax and Km of the enzyme for the substrate (p-nitrophenyl acetate) are 1.948x10(-3) mM/L x dak, and 3.596 mM, respectively. The purification degree of the enzyme was controlled by SDS-PAGE (3-10), which showed two distinct bands. It was determined that the enzyme had activity within the pH range of 4.5-9.5 and that the optimal pH was 7.5. The temperature at which it showed activity was 20-60 degrees C and optimal temperature was 37 degrees C. Molecular weight of CA was found to be 29844 and 61706 Dalton by gel filtration. On the other hand, sulfanilamide and acetazolamide affected the enzyme.     

Characterization of erythrocyte membrane tension for hemolysis prediction in complex flows

Hemolysis is a persistent issue with blood-contacting devices. Many experimental and theoretical contributions over the last few decades have increased insight into the mechanisms of hemolysis in both laminar and turbulent flows, with the ultimate goal of developing a comprehensive, mechanistic hemolysis model. Many models assume that hemolysis scales with a resultant, scalar stress representing all components of the fluid stress tensor. This study critically evaluates this scalar stress hypothesis by calculating the response of the red blood cell membrane to different types of fluid stress (laminar shear and extension, and three turbulent shear and extension cases), each with the same scalar stress. It was found that even though the scalar stress is the same for all cases, membrane tension varied by up to three orders of magnitude. In addition, extensional flow causes constant tension, while tank-treading in shear flow causes periodic tension, with tank-treading frequency varying by three orders of magnitude among the cases. For turbulent flow, tension also depends on eddy size. It is concluded, therefore, that scalar stress alone is inadequate for scaling hemolysis. Fundamental investigations are needed to establish a new index of the fluid stress tensor that provides reliable hemolysis prediction across the wide range of complex flows that occur in cardiovascular devices.     

The organization of the major protein of the human erythrocyte membrane

The enzyme lactoperoxidase was used to catalyse the radioiodination of membrane proteins in intact human erythrocytes and in erythrocyte `ghosts'. Two major proteins of the erythrocyte membrane were isolated after iodination of these two preparations, and the peptide `maps' of each protein so labelled were compared. Peptides from both proteins are labelled in the intact cell. In addition, further mobile peptides derived from one of the proteins are labelled only in the `ghost' preparation. Various sealed `ghost' preparations were also iodinated, lactoperoxidase being present only at either the cytoplasmic or extra-cellular surface of the membrane. The peptide `maps' of protein E (the major membrane protein) labelled in each case were compared. Two discrete sets of labelled peptides were consistently found. One group is obtained when lactoperoxidase is present at the extra-cellular surface and the other group is found when the enzyme is accessible only to the cytoplasmic surface of the membrane. The results support the assumption that the organization of protein E in the membrane of the intact erythrocyte is unaltered on making erythrocyte `ghosts'. They also confirm previous suggestions that both the sialoglycoprotein and protein E extend through the human erythrocyte membrane.     

Structure of neutral glycolipids in bovine thyroid tissue

Four asialo glycolipid fractions have been isolated from bovine thyroid glands. The structures were elucidated by partial hydrolysis, periodate oxidation, permethylation analysis and sequential enzymatic degradation studies. The following structures were identified: GL-1a glucosyl-beta-(1 leads to 1)ceramide; GL-1b galactosyl-beta-(1 leads to 1)ceramide: GL-2 galactosyl-beta-(1 leads to 4)glucosyl-beta-(1 leads to 1)ceramide: GL-3 galactosyl-alpha-(1 leads to 4)galactosyl-beta-(1 leads to 4)glucosyl-beta-(1 leads to 1)-ceramide; GL-4 N-acetylgalactosaminyl-beta-(1 leads to 3)galactosyl-alpha-(1 leads to 4)galactosyl-beta-(1 leads to 4)glucosyl-geta-(1 leads to 1)ceramide.