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.

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.

scholarly journals Biochemical and Genetic Characterization oftrans-2′-Carboxybenzalpyruvate Hydratase-Aldolase from a Phenanthrene-Degrading Nocardioides Strain

1998 ◽  
Vol 180 (4) ◽  
pp. 945-949 ◽  
Author(s):  
Tokuro Iwabuchi ◽  
Shigeaki Harayama
Keyword(s):  
Amino Acid ◽  
Sodium Dodecyl Sulfate ◽  
Amino Acid Sequence ◽  
Gel Electrophoresis ◽  
Genetic Characterization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Degrading Bacterium ◽  
Molecular Masses

ABSTRACT trans-2′-Carboxybenzalpyruvate hydratase-aldolase was purified from a phenanthrene-degrading bacterium,Nocardioides sp. strain KP7, and characterized. The purified enzyme was found to have molecular masses of 38 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography. Thus, the homotrimer of the 38-kDa subunit constituted an active enzyme. The Km andkcat values of this enzyme fortrans-2′-carboxybenzalpyruvate were 50 μM and 13 s−1, respectively.trans-2′-Carboxybenzalpyruvate was transformed to 2-carboxybenzaldehyde and pyruvate by the action of this enzyme. The structural gene for this enzyme was cloned and sequenced; the length of this gene was 996 bp. The deduced amino acid sequence of this enzyme exhibited homology to those oftrans-2′-hydroxybenzalpyruvate hydratase-aldolases fromPseudomonas putida PpG7 and Pseudomonas sp. strain C18.

Investigation of the Peptide Chain of 124 kDa Phytochrome: Localization of Proteolytic Fragments and Epitopes for Monoclonal Antibodies

1986 ◽  
Vol 41 (11-12) ◽  
pp. 993-1000 ◽  
Author(s):  
Rudolf Grimm ◽  
Friedrich Lottspeich ◽  
Hansjörg A. W. Schneider ◽  
Wolfhart Rüdiger
Keyword(s):  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Nucleic Acid ◽  
Amino Acid Sequence ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Amino Terminal ◽  
Differential Reactivity ◽  
Fiber Sheet ◽  
Endogenous Proteases

Abstract Isolated oat phytochrome (124 kDa) was digested with endogenous proteases from oat and with trypsin. Fragments were isolated by SDS polyacrylamide gel electrophoresis (PAGE) blotted onto an activated glass fiber sheet and applied to microsequencing. Comparison of the amino terminal sequences of the fragments with the complete amino acid sequence derived from DNA sequence studies (Hershey et al., Nucleic Acid Res. 13, 8543 - 8560, [1985]) allowed the exact localization of each fragment. The reaction of the various fragments with monoclonal antibodies raised against 114/116 or 124 kDa oat or maize phytochrome were tested by means of Western blotting. Differential reactivity of the localized fragments allowed the localization of epitopes for several antibodies.

scholarly journals Biochemical characterization including amino acid and carbohydrate compositions of canine and human brain Thy-1 antigen

1981 ◽  
Vol 197 (3) ◽  
pp. 629-636 ◽  
Author(s):  
J L McKenzie ◽  
A K Allen ◽  
J W Fabre
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Human Brain ◽  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Antibody Affinity ◽  
Lentil Lectin

Human and canine brain Thy-1 antigens were solubilized in deoxycholate and antigen activity was followed both by conventional absorbed anti-brain xenosera of proven specificity and by mouse monoclonal antibodies to canine and human Thy-1. It is shown that greater than 80% of Thy-1 activity in the dog and man binds to lentil lectin, that the mobility on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of canine and human Thy-1 is identical with that of rat Thy-1 and that the Stokes radius in deoxycholate of canine and human brain Thy-1 is 3.0 nm and 3.25 nm respectively. Both lentil lectin affinity chromatography followed by gel-filtration chromatography on the one hand and monoclonal antibody affinity chromatography on the other gave high degrees of purification of the brain Thy-1 molecule in the dog and man, resulting in single bands staining for both protein and carbohydrate on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (except for a slight contaminant of higher molecular weight staining for protein but not carbohydrate with human Thy-1 purified by lentil lectin and gel-filtration chromatography). Analysis of canine and human brain Thy-1 purified by monoclonal antibody affinity chromatography with additional gel filtration through Sephadex G-200 showed that these molecules had respectively 38% and 36% carbohydrate. The amino acid and carbohydrate compositions were similar to those previously reported for Thy-1 of the rat and mouse, the main point of interest being the presence in canine and human brain Thy-1 of N-acetylgalactosamine, which has been reported in rat and mouse brain Thy-1 but not in Thy-1 from other tissues.

scholarly journals Structural properties of homogeneous protein disulphide-isomerase from bovine liver purified by a rapid high-yielding procedure

1983 ◽  
Vol 213 (1) ◽  
pp. 225-234 ◽  
Author(s):  
N Lambert ◽  
R B Freedman
Keyword(s):  
Amino Acid ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Bovine Liver ◽  
Polyacrylamide Gel ◽  
Protein Disulphide Isomerase

Protein disulphide-isomerase from bovine liver was purified to homogeneity as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, two-dimensional electrophoresis and N-terminal amino acid analysis. The preparative procedure, a modification of that of Carmichael, Morin & Dixon [(1977) J. Biol. Chem. 252, 7163-7167], is much faster and higher-yielding than previous procedures, and the final purified material is of higher specific activity. The enzyme has Mr 57 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, both in the presence and in the absence of thiol compounds. Gel-filtration studies on Sephadex G-200 indicate an Mr of 107 000, suggesting that the native enzyme is a homodimer with no interchain disulphide bonds. Ultracentrifugation studies give a sedimentation coefficient of 3.5S, implying that the enzyme sediments as the monomer. The isoelectric point, in the presence of 8 M-urea, is 4.2, and some microheterogeneity is detectable. The amino acid composition is comparable with previous analyses of this enzyme from bovine liver and of other preparations of thiol:protein disulphide oxidoreductases whose relation to protein disulphide-isomerase has been controversial. The enzyme contains a very high proportion of Glx + Asx residues (27%). The N-terminal residue is His. The pure enzyme has a very small carbohydrate content, determined as 0.5-1.0% by the phenol/H2SO4 assay. Unless specific steps are taken to remove it, the purified enzyme contains a small amount (5 mol/mol of enzyme) of Triton X-100 carried through the purification.

Normal Mature Human Enamel Protein

1979 ◽  
Vol 58 (2_suppl) ◽  
pp. 986-987 ◽  
Author(s):  
A. Belcourt
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Gel Electrophoresis ◽  
Protein Concentration ◽  
Polyacrylamide Gel Electrophoresis ◽  
Organic Matrix ◽  
Low Molecular Weight ◽  
Human Enamel ◽  
Weak Density ◽  
Enamel Protein

Pure enamel was prepared using an original microdissection technic. Protein concentration was 375 μg per gram of enamel. Polyacrylamide gel electrophoresis showed a single fast-migrating zone containing a thin double band. Ultracentrifugation studies suggested that the proteins were of low molecular weight or of weak density. Absorption spectra showed a strong absorbance at 260nm. Amino acid analyses yielded a composition of 25% Gly, 13.5% Glu, 11% Ser, 11% Pro, 2% Cys and 2% Hyp. A glucidic content of 15% was estimated and glucose, galactose, mannose and fucose were identified. The organic matrix of enamel seemed to be constituted of two major glycoproteins probably fibrous but different from keratin.

scholarly journals The purification and properties of the second component of human complement

1978 ◽  
Vol 171 (1) ◽  
pp. 99-107 ◽  
Author(s):  
M A Kerr ◽  
R R Porter
Keyword(s):  
Amino Acid ◽  
Polypeptide Chain ◽  
Alternative Pathway ◽  
Polyacrylamide Gel Electrophoresis ◽  
Complement Factor ◽  
Human Complement ◽  
Terminal Amino Acid ◽  
Factor B ◽  
Antigenic Properties ◽  
Terminal Amino

The second component of human complement (C2) was purified by a combination of euglobulin precipitation, ion-exchange chromatography, (NH4)2SO4 precipitation and affinity chromatography. The final product was homogeneous by the criterion of polyacrylamide-gel electrophoresis and represents a purification of about 4000-fold from serum with 15-20% yield. Component C2 comprises a single carbohydrate-containing polypeptide chain, with an apparent mol.wt. of 102000; alanine is the N-terminal amino acid. The molecule is rapidly cleaved by activated subcomponent C1s with the loss of haemolytic activity to yield two fragments with apparent mol.wts. of 74000 and 34000. These fragments are not linked by disulphide bonds and can be easily separated. A second protein isolated during the purification of component C2 was identified by its haemolytic and antigenic properties as complement Factor B, the protein serving an analogous function to component C2 in the alternative pathway. The protein, which is also a single carbohydrate-containing polypeptide chain, has an apparent mol.wt. of 95000 and threonine as N-terminal amino acid. The amino acid analyses of component C2 and Factor B are compared.

Subunit studies of coupling factor 1 of bean chloroplasts

1976 ◽  
Vol 54 (5) ◽  
pp. 481-487 ◽  
Author(s):  
M. P. Silvanovich ◽  
R. D. Hill
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Coupling Factor ◽  
Leaf Extracts ◽  
Molecular Weights ◽  
Major Species ◽  
Chloroplast Coupling Factor ◽  
Coupling Factors

A bean chloroplast coupling factor (CF1) with latent Ca2+-dependent ATPase activity was studied. Immunodiffusion of bean (Phaseolus vulgaris) chloroplast and etioplast coupling factors and spinach coupling factor against antiserum to spinach coupling factor showed partial identity of the bean coupling factor with that of spinach. An immunoelectrophoretic comparison, under dissociating conditions, of bean leaf extracts and spinach extracts containing CF1 subunits (as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis) gave identical results for both extracts. At least six distinct polypeptide species were found. The major species had molecular weights of 42 000, 59 000 and 63 000 daltons. Amino acid analysis of electrophoretically purified bean CF1 gave results similar to those published for spinach CF1.

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