scholarly journals Abnormal blood-group-Ss-active sialoglycoproteins in the membrane of Miltenberger class III, IV and V human erythrocytes

1979 ◽  
Vol 183 (2) ◽  
pp. 193-203 ◽  
Author(s):  
D J Anstee ◽  
W J Mawby ◽  
M J A Tanner
Keyword(s):  
Blood Group ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Normal Blood ◽  
Crossing Over ◽  
Class Iii ◽  
Class V ◽  
Unequal Crossing Over

1. We have studied the inherited changes occurring in the sialoglycoproteins of membranes from erythrocytes of type Miltenberger Class III (Mi.III), Miltenberger Class IV (Mi.IV) and Miltenberger Class V (Mi.V) by using sodium dodecyl sulphate/polyacrylamide gel electrophoresis and lactoperoxidase radioiodination. 2. Mi.III erythrocytes lack the normal blood-group-Ss-active sialoglycoprotein but contain an unusual s-active sialoglycoprotein of higher apparent molecular weight. A similar abnormal S-active sialoglycoprotein appears to occur in Mi.IV erythrocytes. 3. The Mi.V condition is associated with the hemizygous absence of both the normal blood-group-MN-active sialoglycoprotein and the normal Ss-active sialoglycorprotein. However, a new sialoglycoprotein component is present in these cells that has properties characteristic of both the MN-active and Ss-active sialoglycoproteins. 4. Our results suggest that the new sialoglycorportein present in Mi.V erythrocytes is a hybrid of the normal MN sialoglycoprotein and an s-active sialoglycoprotein that has properties similar to the s-active sialoglycoprotein found in Mi.III erythrocytes. We suggest that the unusual Mi.V sialoglycoprotein is derived from chromosomal misalignment with unequal crossing-over between the genes for the MN- and Ss-active sialoglycoproteins in a manner similar to that which gives rise to haemoglobin Lepore. 5. Further studies of S-s-erythrocytes confirm that these cells lack normal Ss-active sialoglycoprotein, but contain an unusual component that shows some of the properties of the normal Ss-active sialoglycoprotein. 6. Analysis of erythrocytes of type Mk/Mi.III confirms that, in addition to the known hemizygous lack of the MN-active sialoglycoprotein, the Mk condition is also associated with a loss of the Ss-active sialoglycoprotein. 7. In order to facilitate discussion of the complex changes that occur in these variant erythrocytes, a new unified nomenclature is used for the erythrocyte sialoglycoproteins.

Variation in goat fibre protein

1989 ◽  
Vol 40 (3) ◽  
pp. 675 ◽  
Author(s):  
DJ Tucker ◽  
AHF Hudson ◽  
A Laudani ◽  
RC Marshall ◽  
DE Rivett
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Wool Fibre ◽  
Two Dimensional ◽  
Protein Patterns ◽  
Cashmere Goats

The proteins from a range of cashmere, mohair, angoratcashmere crossbred and wool fibre samples were extracted at pH 8 with 8 M urea containing dithiothreitol, and were then radiolabelled by S-carboxymethylation using iodo(2-14C) acetate. The proteins from each sample were examined by two dimensional polyacrylamide gel electrophoresis in which the separation in the first dimension was according to charge at pH 8.9 and in the second dimension according to apparent molecular weight in the presence of sodium dodecyl sulfate. After electrophoresis the proteins were detected by fluorography. Protein differences in keratin samples from some individual goats existed, although the overall protein patterns were similar. None of the differences were consistent with any one goat fibre type. The protein patterns obtained for fibre samples from individual cashmere goats showed some differences when compared to those found for commercial blends from the same country of origin, indicating that blending can mask any animal-to-animal variation. While the electrophoretic technique does not unequivocally distinguish between cashmere, mohair and angora/cashmere crossbred fibres it does differentiate between wool and goat fibres.

Sugar profiling proves that human serum erythropoietin differs from recombinant human erythropoietin

Blood ◽  
2001 ◽  
Vol 98 (13) ◽  
pp. 3626-3634 ◽  
Author(s):  
Venke Skibeli ◽  
Gro Nissen-Lie ◽  
Peter Torjesen
Keyword(s):  
Human Serum ◽  
Gel Electrophoresis ◽  
High Performance ◽  
Magnetic Beads ◽  
Direct Method ◽  
Polyacrylamide Gel Electrophoresis ◽  
Broad Band ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Two Dimensional Gel Electrophoresis

Abstract Erythropoietin (EPO) from sera obtained from anemic patients was successfully isolated using magnetic beads coated with a human EPO (hEPO)–specific antibody. Human serum EPO emerged as a broad band after sodium dodecyl sulfate–polyacrylamide gel electrophoresis, with an apparent molecular weight slightly smaller than that of recombinant hEPO (rhEPO). The bandwidth corresponded with microheterogeneity because of extensive glycosylation. Two-dimensional gel electrophoresis revealing several different glycoforms confirmed the heterogeneity of circulating hEPO. The immobilized anti-hEPO antibody was capable of binding a representative selection of rhEPO glycoforms. This was shown by comparing normal-phase high-performance liquid chromatography profiles of oligosaccharides released from rhEPO with oligosaccharides released from rhEPO after isolation with hEPO-specific magnetic beads. Charge analysis demonstrated that human serum EPO contained only mono-, di-, and tri-acidic oligosaccharides and lacked the tetra-acidic structures present in the glycans from rhEPO. Determination of charge state after treatment of human serum EPO with Arthrobacter ureafaciens sialidase showed that the acidity of the oligosaccharide structures was caused by sialic acids. The sugar profiles of human serum EPO, describing both neutral and charged sugar, appeared significantly different from the profiles of rhEPO. The detection of glycan structural discrepancies between human serum EPO and rhEPO by sugar profiling may be significant for diagnosing pathologic conditions, maintaining pharmaceutical quality control, and establishing a direct method to detect the misuse of rhEPO in sports.

scholarly journals Human plasma alpha 2-macroglobulin and von Willebrand factor possess covalently linked ABO(H) blood group antigens in subjects with corresponding ABO phenotype

Blood ◽  
1993 ◽  
Vol 82 (2) ◽  
pp. 663-668
Author(s):  
T Matsui ◽  
Y Fujimura ◽  
S Nishida ◽  
K Titani
Keyword(s):  
Blood Group ◽  
Von Willebrand Factor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Enzyme Linked Immunosorbent Assay ◽  
Blood Group Antigens ◽  
Ulex Europaeus ◽  
Von Willebrand ◽  
Willebrand Factor

We recently identified ABO(H) blood group structures in Asn-linked sugar chains of human von Willebrand factor (vWF) purified from factor VIII concentrates (J Biol Chem 267:8723, 1992). We surveyed plasma glycoproteins carrying ABO(H) blood group antigens by Western blotting analysis and sandwich enzyme-linked immunosorbent assay using blood group-specific monoclonal antibodies (MoAbs) and a lectin. Two major plasma proteins showing apparent molecular weight of about 180 Kd and 270 Kd by sodium dodecyl sulfate polyacrylamide gel electrophoresis reacted with blood group-specific MoAbs and Ulex europaeus lectin I in accordance with donor blood group. Direct sequence analysis of the protein bands showed their identity with the N-terminal sequences of alpha 2-macroglobulin (alpha 2M) and vWF, respectively. The two bands also reacted with anti-alpha 2M and anti-vWF antibodies. The alpha 2M and vWF prepared from plasma by immunoprecipitation showed the appropriate blood group antigenicity. After incubation with endoglycosidase F, both alpha 2M and vWF lost almost all reactivity with anti-blood group reagents. About 90% of plasma vWF, but only approximately 10% of alpha 2M, was immunoprecipitated with anti-blood group antibody. These results indicate that at least two plasma glycoproteins, vWF and alpha 2M, possess Asn-linked ABO(H) blood group antigens in normal individuals with corresponding ABO phenotype. Therefore, ABO(H) blood group antigens in plasma glycoproteins should be considered during preparation of plasma materials for therapeutic use.

scholarly journals Genetic variants involving the major membrane sialoglycoprotein of human erythrocytes. Studies on erythrocytes of type Mk, Miltenberger class V and Mg

1978 ◽  
Vol 175 (1) ◽  
pp. 149-157 ◽  
Author(s):  
D J Anstee ◽  
M J A Tanner
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Blood Group ◽  
Genetic Variants ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Membrane Component ◽  
Class V ◽  
Normal Cells ◽  
Antigenic Properties

1. Membranes from erythrocytes heterozygous for the Mk and Miltenberger Class V (Mi.V) condition and membranes from erythrocytes homozygous for the Mg condition were studied by polyacrylamide-gel electrophoresis by using the periodate/Schiff stain binding of radioiodinated lectins and labelling with lactoperoxidase. 2. Both the Mk and Mi.V conditions are associated with a decreased content of the major blood-group-MN-active sialoglycoprotein. 3. An unusual blood-group-M-active membrane component was found in Mi.V cells of appropriate genotype. No comparably component was found in Mk erythrocytes. 4. The Mg antigen appears to result from a modification of the MN-active sialoglycoprotein found in normal cells. Our results suggest that the Mg sialoglycoprotein contains fewer sialotetrasaccharides than does the normal sialglycoprotein. This may result from changes in the amino acid sequence of the protein. 5. The results are discussed in relation to differences in the antigenic properties of Mk, Mi.V and Mg cells and their possible influence on the structure of the surface of each of these cells.

Products of mitochondrial protein synthesis in Tetrahymena

1979 ◽  
Vol 57 (4) ◽  
pp. 314-320 ◽  
Author(s):  
Paul G. Young ◽  
Neil P. Hunter
Keyword(s):  
Protein Synthesis ◽  
Gel Electrophoresis ◽  
Trichloroacetic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mitochondrial Protein ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Mitochondrial Proteins ◽  
Mitochondrial Protein Synthesis ◽  
Chloroform Methanol

The products of mitochondrial protein synthesis have been investigated in Tetrahymena after labelling with [35S]methionine in the presence of cycloheximide. The labelled proteins were analyzed by sodium dodecyl sulfate slab polyacrylamide gel electrophoresis. We have identified 13 electrophoretically discrete bands as well as 4 other bands with a more variable occurrence. These proteins ranged in apparent molecular weight from 8100 to 57 500. The cycloheximide-resistant incorporation could be blocked with chloramphenicol. The mitochondrial proteins appeared to be in a disaggregated state and were stable to agents such as trichloroacetic acid (hot or cold) and chloroform–methanol. The pattern of proteins was similar following labelling times ranging from 30 min to 3 h.

scholarly journals Purification of the Thy-1 molecule, a major cell-surface glycoprotein of rat thymocytes

1975 ◽  
Vol 151 (3) ◽  
pp. 685-697 ◽  
Author(s):  
M Letarte-Muirhead ◽  
A N Barclay ◽  
A F Williams
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Surface Glycoprotein ◽  
Cell Surface Glycoprotein ◽  
Lentil Lectin ◽  
Polypeptide Chains

The Thy-1-molecule, which was identified by its antigenic activities, has been purified from rat thymocytes. The purification involved preparation of crude membranes and solubilization in deoxycholate, followed by gel filtration and affinity chromatography on antibody or lectin columns. In all cases the purified molecule was a glycoprotein that did not form higher polymers and was not associated with other polypeptide chains. The Thy-1 glycoprotein could be found in two forms, one binding to lentil lectin, the other not. Both forms had the same detectable antigens and were of a similar but not identical size. After sodium dodecyl sulphate-polyacrylamide-gel electrophoresis the apparent molecular weight of Thy-1 binding to lentil lectin was 25 000, whereas that not binding to the lectin was 27 000, with heterogeneity towards forms of apparently higher molecular weight.

A Sensitive Peroxidase Staining Immunoblotting Method for Measuring Total Protein S in Human Plasma

1993 ◽  
Vol 69 (04) ◽  
pp. 331-334 ◽  
Author(s):  
Xiangan Li ◽  
Kaoru Hatanaka ◽  
Ling Guo ◽  
Motoo Tsushima ◽  
Yukihiko Kitamura ◽  
...  
Keyword(s):  
Human Plasma ◽  
Total Protein ◽  
Gel Electrophoresis ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Protein S ◽  
Polyacrylamide Gel ◽  
Free Form

SummaryIn plasma, protein S is found in its free form and as a complex with C4b-binding protein. After 125I-protein S was added tonormal human plasma and applied to SDS-8% polyacrylamide gel electrophoresis, the autoradiogram of the gel showed only one single band at free protein S position. Applying this evidence, we have developed a peroxidase staining Western Blotting method to quantitate total protein S in human plasma which consists of sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by blotting to nitrocellulose membrane and a sensitive avidin-biotinylated peroxidase staining method (ABC technique). The measurement of protein S by the immunoblotting was reproducible and the coefficient of variation was 7%. As little as 1 ng of protein S could be detected. C4b-binding protein did not affect the measurement of protein S. Compared to other immunoassays, this peroxidase staining immunoblotting method has the advantage of directly estimating the apparent molecular weight of protein of interest, eliminating nonspecific stain and having high sensitivity without using radioisotope.

scholarly journals Subunit structure of Vicia graminea anti-(blood-group N) lectin

1980 ◽  
Vol 189 (1) ◽  
pp. 185-188 ◽  
Author(s):  
M J Prigent ◽  
R Bourrillon
Keyword(s):  
Blood Group ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Subunit Structure ◽  
Terminal Sequence ◽  
Guanidinium Chloride ◽  
Molecular Weights ◽  
Covalently Linked

The subunit of the Vicia graminea lectin with blood-group-N specificity was examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration in 6M-guanidinium chloride, and its molecular weights was found to be 25 000. The unique N-terminal sequence fof the first nine residues of the lectin confirmed that Vicia lectin consists of four identical chains non-covalently linked. Finally the microheterogeneity of the lectin shown by analytical isoelectric focusing is discussed.

Cell surface protein of Salmonella typhimurium

1975 ◽  
Vol 21 (7) ◽  
pp. 1132-1136 ◽  
Author(s):  
S. Kabir
Keyword(s):  
Cell Surface ◽  
Salmonella Typhimurium ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Protein ◽  
Apparent Molecular Weight ◽  
Major Protein ◽  
Cell Surface Protein ◽  
Exterior Surface

Lactoperoxidase-catalyzed radioiodination with Na125I was performed both on intact Salmonella typhimurium 1195 and on ghost membrane isolated from the same bacterial strain. Ghost membrane was also prepared from radioiodinated whole bacteria. The labelled proteins from both these ghost membrane preparations were compared by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate to identify the cell surface protein. From the results obtained it was concluded that one major protein with an apparent molecular weight of 12 000 – 13 000 was exposed on the exterior surface of the ghost membrane.

scholarly journals Human plasma alpha 2-macroglobulin and von Willebrand factor possess covalently linked ABO(H) blood group antigens in subjects with corresponding ABO phenotype

Blood ◽  
1993 ◽  
Vol 82 (2) ◽  
pp. 663-668 ◽  
Author(s):  
T Matsui ◽  
Y Fujimura ◽  
S Nishida ◽  
K Titani
Keyword(s):  
Blood Group ◽  
Von Willebrand Factor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Enzyme Linked Immunosorbent Assay ◽  
Blood Group Antigens ◽  
Ulex Europaeus ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We recently identified ABO(H) blood group structures in Asn-linked sugar chains of human von Willebrand factor (vWF) purified from factor VIII concentrates (J Biol Chem 267:8723, 1992). We surveyed plasma glycoproteins carrying ABO(H) blood group antigens by Western blotting analysis and sandwich enzyme-linked immunosorbent assay using blood group-specific monoclonal antibodies (MoAbs) and a lectin. Two major plasma proteins showing apparent molecular weight of about 180 Kd and 270 Kd by sodium dodecyl sulfate polyacrylamide gel electrophoresis reacted with blood group-specific MoAbs and Ulex europaeus lectin I in accordance with donor blood group. Direct sequence analysis of the protein bands showed their identity with the N-terminal sequences of alpha 2-macroglobulin (alpha 2M) and vWF, respectively. The two bands also reacted with anti-alpha 2M and anti-vWF antibodies. The alpha 2M and vWF prepared from plasma by immunoprecipitation showed the appropriate blood group antigenicity. After incubation with endoglycosidase F, both alpha 2M and vWF lost almost all reactivity with anti-blood group reagents. About 90% of plasma vWF, but only approximately 10% of alpha 2M, was immunoprecipitated with anti-blood group antibody. These results indicate that at least two plasma glycoproteins, vWF and alpha 2M, possess Asn-linked ABO(H) blood group antigens in normal individuals with corresponding ABO phenotype. Therefore, ABO(H) blood group antigens in plasma glycoproteins should be considered during preparation of plasma materials for therapeutic use.

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