scholarly journals The anion-transport protein of the human erythrocyte membrane. Studies on fragments produced by pepsin digestion

1979 ◽  
Vol 183 (2) ◽  
pp. 417-427 ◽  
Author(s):  
M J A Tanner ◽  
D G Williams ◽  
D Kyle
Keyword(s):  
Acetic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Anion Transport ◽  
Transport Protein ◽  
Erythrocyte Membranes ◽  
Major Site ◽  
Neutral Ph ◽  
C Terminus ◽  
Peptide Maps

We have studied the fragmentation by pepsin in 1 M-acetic acid of the erythrocyte anion-transport protein in erythrocyte membranes. The location of the fragments obtained was determined by radioiodinating the protein with the use of lactoperoxidase, and identifying the labelled peptides obtained in peptide “maps” of thermolysin digests of the fragments. Three of the fragments were found to be related overlapping products, and shared a common C-terminus. The major site of pepsin cleavage leading to the C-termini of these fragments was shown to be close to the major site of extracellular cleavage of the protein by proteinases active at a neutral pH. Another two fragments were isolated and shown to be derived from the C-terminal portion of the protein. No well-defined large radioactive fragments of the protein were solubilized from the membrane by pepsin in 1 M-acetic acid, the bulk of the radioactivity attributable to the anion transport protein being recovered in very small fragments that could not be resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Our results suggest that the polypeptide chain of the anion-transport protein emerges at the extracellular face of the membrane 8000-13000 daltons on the N-terminal side of the major site of extracellular cleavage of the protein by proteinases that are active at a neutral pH.

Conformation and stability of the anion transport protein of human erythrocyte membranes

Biochemistry ◽  
1985 ◽  
Vol 24 (12) ◽  
pp. 2843-2848 ◽  
Author(s):  
Kimio Oikawa ◽  
Debra M. Lieberman ◽  
Reinhart A. F. Reithmeier
Keyword(s):  
Human Erythrocyte ◽  
Anion Transport ◽  
Transport Protein ◽  
Erythrocyte Membranes ◽  
Human Erythrocyte Membranes

scholarly journals Affinity purification and subunit structures of β-N-acetylhexosaminidases A and B from boar epididymis

1984 ◽  
Vol 219 (3) ◽  
pp. 1009-1015 ◽  
Author(s):  
H C Parkes ◽  
J L Stirling ◽  
P Calvo
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Gradient Gel Electrophoresis ◽  
Sepharose 4B ◽  
Electrophoretic Transfer ◽  
Peptide Maps

beta-N-Acetylhexosaminidase from boar epididymis was separated into two forms, A and B, on DEAE-cellulose. Both these forms were excluded from Sepharose S-200 and had apparent Mr values of 510 000 on gradient gel electrophoresis under non-denaturing conditions. Affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-beta-D-glucopyranosylam ine coupled to CNBr-activated Sepharose 4B was used to separate and purify beta-N-acetylhexosaminidases A and B that had specific activities of 115 and 380 mumol/min per mg of protein respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of denatured beta-N-acetylhexosaminidase A gave a single major component of Mr 67 000. beta-N-Acetylhexosaminidase B also had this component, and in addition had polypeptides of Mr 29 000 and 26 000. All these polypeptides were glycosylated. Antiserum to the B form precipitated form A from solution and reacted with the 67 000-Mr component or form A after electrophoretic transfer from sodium dodecyl sulphate/polyacrylamide gels to nitrocellulose sheets. The 67 000-Mr components of forms A and B yielded identical peptide maps when digested with Staphylococcus aureus V8 proteinase, and the 29 000-Mr and 26 000-Mr components in form B may be related to the 67 000-Mr polypeptide.

scholarly journals The inhibition of platelet cyclo-oxygenase by aspirin is associated with the acetylation of a 72kDa polypeptide in the intracellular membranes

1984 ◽  
Vol 223 (1) ◽  
pp. 105-111 ◽  
Author(s):  
N Hack ◽  
F Carey ◽  
N Crawford
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Membrane System ◽  
Particulate Material ◽  
Intracellular Membrane ◽  
Membrane Structures ◽  
Major Site ◽  
Intracellular Membranes ◽  
Oxygenase Activity ◽  
Radioactive Labelling

Previous studies by Roth & Majerus [J. Clin. Invest. (1975) 56, 624-632] showed that exposure of platelets to [acetyl-14C]aspirin resulted in the radioactive labelling of three polypeptides, two of which were in the cytosol and not saturable, whilst the third was located in particulate material, and was saturated at 30 microM-aspirin. By using high voltage free flow electrophoresis to separate a platelet mixed membrane fraction into highly purified surface and intracellular membrane subfractions, we have confirmed that the major polypeptide acetylated after exposing whole platelets to [acetyl-14C]aspirin is almost exclusively associated with intracellular membrane structures. We have shown previously that these intracellular membranes are the major site for prostanoid biosynthesis [Carey, Menashi & Crawford (1982) Biochem. J. 204, 847-851] and in the present study the extent of the radioactive labelling correlated well with inhibition of the cyclo-oxygenase activity in these intracellular membranes. In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis the [14C]acetylated component, which appears to be a dimer, migrates with a mobility corresponding to 72kDa. Although cyclo-oxygenase is inhibited, there is no discernible radioactive labelling when the platelets are exposed to aromatic-ring-labelled [14C]aspirin. We suggest that the site or sites for aspirin acetylation and cyclo-oxygenase activity are structurally associated in the platelet's intracellular membranes referred to by electron microscopists as the dense tubular membrane system.

scholarly journals Proinflammatory cytokines induce accumulation of glypican-1-derived heparan sulfate and the C-terminal fragment of β-cleaved APP in autophagosomes of dividing neuronal cells

Glycobiology ◽  
2020 ◽  
Vol 30 (8) ◽  
pp. 539-549
Author(s):  
Fang Cheng ◽  
Lars-Åke Fransson ◽  
Katrin Mani
Keyword(s):  
Heparan Sulfate ◽  
Proinflammatory Cytokines ◽  
Polyacrylamide Gel Electrophoresis ◽  
Neuronal Cells ◽  
Sodium Dodecyl ◽  
Terminal Fragment ◽  
C Terminus ◽  
Tnf Α ◽  
Sds Page ◽  
N Terminus

Abstract Proinflammatory cytokines stimulate expression of β-secretase, which increases processing of amyloid precursor protein (APP), ultimately leading to the deposition of amyloid beta (Aβ). The N-terminal domain of β-cleaved APP supports Cu/NO-dependent release of heparan sulfate (HS) from the glypican-1 (Gpc-1) proteoglycan. HS is an inhibitor of β-secretase, thereby constituting a regulatory, negative feedback loop. Here, we have investigated the effect of the proinflammatory cytokines TNF-α, IL-1β and IL-6 on the interplay between APP processing and release of HS from Gpc-1 in neuronal cells. We have used deconvolution immunofluorescence microscopy and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and a panel of monoclonal/polyclonal antibodies recognizing the released HS, the N-terminus of Aβ, Aβ, the C-terminus of APP and the autophagosome marker LC3 as well as the chemical lysosome marker LysoTrackerRed (LTR). We repeatedly found that N2a neuroblastoma cells and human neural stem cells grown in the presence of the cytokines developed large cytoplasmic clusters, which stained positive for HS, the N-terminus of Aβ, Aβ, the C-terminus of APP, LC3 and LTR, indicating accumulation of HS and APP/APP degradation products in enlarged autophagosomes/lysosomes. The SDS-PAGE of immunoisolates obtained from TNF-α-treated N2a cells by using anti-C-terminus of APP revealed the presence of SDS-stable complexes between HS and the C-terminal fragment of β-cleaved APP (βCTF) migrating in the range 10–18 kDa. Clustered accumulation of βCTF disappeared when HS release was prevented and slightly enhanced when HS release was increased. Hence, when proinflammatory cytokines induce increased processing of APP, inhibition of β-secretase by HS is insufficient, which may lead to the impaired autophagosomal degradation.

scholarly journals Comparative study of glycophorin A derived O-glycans from human Cad, Sd(a+) and Sd(a-) erythrocytes

1985 ◽  
Vol 232 (3) ◽  
pp. 813-818 ◽  
Author(s):  
D Blanchard ◽  
C Capon ◽  
Y Leroy ◽  
J P Cartron ◽  
B Fournet
Keyword(s):  
Blood Group ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Red Cells ◽  
Erythrocyte Membranes ◽  
Glycophorin A ◽  
Methylation Analysis ◽  
Dolichos Biflorus ◽  
Major Acid

Glycophorin A was purified from the erythrocyte membranes of blood group Cad, Sd(a+) and Sd(a-) donors and the oligosaccharide alditols, obtained after alkaline borohydride degradation, separated by h.p.l.c. on an alkylamine silica gel column, were characterized by sugar analysis. Structure determination of the major acid components by methylation analysis, g.l.c.-m.s. and 1H-n.m.r. indicated that the three blood group Cad red cells under study (samples Cad., Bui. and Des.) carry the same pentasaccharide GalNAc(beta 1-4)[NeuAc(alpha 2-3)]Gal(beta 1-3)[NeuAc(alpha 2-6)]GalNAc -ol(Cad determinant) but in different amounts. This pentasaccharide, however, was absent from glycophorin A of Sd(a+) and Sd (a-) donors, suggesting that the Sda determinant is not associated with glycophorins. It was calculated that glycophorin A from the original Cad donor (Cad.) carries about 12 O-glycosidically linked pentasaccharide chains per molecule whereas only 2-3 of these chains were present in the samples from the two other unrelated Cad individuals (Bui. and Des.) It is well known from quantitative agglutination studies that the proportion of red cells which can be agglutinated by the Dolichos biflorus lectin varies from one Cad blood sample to another. Some are completely agglutinated (Cad. donor) whereas others are only partially agglutinated (Bui. and Des. donors) suggesting that some red cells might not carry the Cad determinants. From the results presented above and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis studies it is suggested that Cad red cells from Bui. and Des. do not carry a mixture of glycophorin A molecules with or without the Cad pentasaccharides but a spectrum of glycoprotein molecules with varying amounts of Cad determinants.

scholarly journals Glycophorin A in two patients with congenital dyserythropoietic anemia type I and type II is partly unglycosylated.

2000 ◽  
Vol 47 (3) ◽  
pp. 773-779 ◽  
Author(s):  
E Zdebska ◽  
M Adamczyk-Popławska ◽  
J Kościelak
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Glycosylation Site ◽  
Erythrocyte Membranes ◽  
Glycophorin A ◽  
Type I ◽  
Type Ii ◽  
Congenital Dyserythropoietic Anemia ◽  
Significant Deficit ◽  
Preliminary Extraction

Glycophorins A from erythrocyte membranes of two patients with congenital dyserythropoietic anemia type I and type II (CDA type I and II) were analyzed for carbohydrate molar composition employing a modification of the recently published method that allowed simultaneous determination of carbohydrates and protein in electrophoretic bands of glycoproteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Zdebska & Kościelak, 1999, Anal Biochem., 275, 171-179). The modification involved a preliminary extraction of erythrocyte membranes with aqueous phenol, subsequent electrophoresis and analysis of the extracted glycophorins rather than electrophoresis and analysis of the glycophorin from intact erythrocyte membranes. The results showed a large deficit of N-acetylgalactosamine, galactose, and sialic acid residues in glycophorin A from patients with CDA type I and type II amounting to about 45% and 55%, respectively. The results strongly suggest that glycophorin A in these patients is partly unglycosylated with respect to O-linked glycans. In addition, glycophorin A from erythrocytes of a patient with CDA II but not CDA I exhibited a significant deficit of mannose and N-acetylglucosamine suggesting that its N-glycosylation site was also partly unglycosylated.

Expression of Na(+)-K(+)-ATPase alpha 1- and alpha 3-isoforms in adult and neonatal ferret hearts

1992 ◽  
Vol 263 (5) ◽  
pp. H1430-H1436
Author(s):  
Y. C. Ng ◽  
C. B. Book
Keyword(s):  
Peptide Mapping ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Right Atrial ◽  
Neonatal Heart ◽  
Ventricular Tissue ◽  
Left And Right ◽  
Peptide Maps ◽  
Ferret Heart

We have demonstrated previously that in adult ferret heart two alpha-subunit isoforms of the Na(+)-K(+)-ATPase, alpha(+) and alpha, are expressed. The alpha(+)-isoform may comprise either alpha 2-, or alpha 3-, or both isoforms. The present studies further characterize the alpha(+)-isoform. The alpha(+)-isoform of ferret heart did not react with an alpha 2-specific monoclonal antibody, but rather with two different alpha 3-specific polyclonal antibodies. Electrophoretic mobility of the alpha(+)-isoform in sodium dodecyl sulfate polyacrylamide gel electrophoresis is slower than that of the alpha 2-isoform, but similar to that of the alpha 3-isoform. Limited proteolytic peptide mapping was performed using Staphylococcus aureus V8. Proteolytic fragments were then immunostained with an alpha 3-specific antibody. The peptide maps of ferret heart alpha(+)-isoform and rat brain alpha 3-isoform were identical, as were those of ferret heart alpha(+)-isoform and ferret brain alpha 3-isoform. These results indicate that the alpha(+)-isoform of ferret heart is an alpha 3-isoform. During postnatal development, the same isoforms expressed in the adult ferret heart (alpha 1 and alpha 3), were also expressed in neonatal heart. In adult or neonatal heart alpha 2-isoform was not detectable. Relative abundances of the isoforms in ventricular and atrial tissues differed. Compared with ventricular tissue, left and right atrial tissues expressed much less alpha 3 than alpha 1. It is concluded that, unlike rat heart, alpha 1- and alpha 3-isoforms are expressed in adult ferret heart.(ABSTRACT TRUNCATED AT 250 WORDS)

Tropomyosin isoform 5b is expressed in human erythrocytes: implications of tropomodulin-TM5 or tropomodulin-TM5b complexes in the protofilament and hexagonal organization of membrane skeletons

Blood ◽  
2000 ◽  
Vol 95 (4) ◽  
pp. 1473-1480 ◽  
Author(s):  
Lanping Amy Sung ◽  
Ke-Ming Gao ◽  
Leland J. Yee ◽  
Constance J. Temm-Grove ◽  
David M. Helfman ◽  
...  
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Membrane Skeleton ◽  
Actin Binding ◽  
Erythrocyte Membranes ◽  
Erythroid Terminal Differentiation ◽  
Common Features ◽  
Capping Protein ◽  
Major Species ◽  
Molecular Ruler

The human erythrocyte membrane skeleton consists of hexagonal lattices with junctional complexes containing F-actin protofilaments of approximately 33-37 nm in length. We hypothesize that complexes formed by tropomodulin, a globular capping protein at the pointed end of actin filaments, and tropomyosin (TM), a rod-like molecule of approximately 33-35 nm, may contribute to the formation of protofilaments. We have previously cloned the human tropomodulin complementary DNA and identified human TM isoform 5 (hTM5), a product of theγ-TM gene, as one of the major TM isoforms in erythrocytes. We now identify TM5b, a product of the -TM gene, to be the second major TM isoform. TM5a, the alternatively spliced isoform of the-TM gene, which differs by 1 exon and has a weaker actin-binding affinity, however, is not present. TM4, encoded by the δ-TM gene, is not present either. In sodium dodecyl sulfate–polyacrylamide gel electrophoresis, hTM5 comigrated with the slower TM major species in erythrocyte membranes, and hTM5b comigrated with the faster TM major species. TM5b, like TM5, binds strongly to tropomodulin, more so than other TM isoforms. The 2 major TM isoforms, therefore, share several common features: They have 248 residues, are approximately 33-35 nm long, and have high affinities toward F-actin and tropomodulin. These common features may be the key to the mechanism by which protofilaments are formed. Tropomodulin-TM5 or tropomodulin-TM5b complexes may stabilize F-actin in segments of approximately 33-37 nm during erythroid terminal differentiation and may, therefore, function as a molecular ruler. TM5 and TM5b further define the hexagonal geometry of the skeletal network and allow actin-regulatory functions of TMs to be modulated by tropomodulin.

scholarly journals Dog and human acid β-d-galactosidases are structurally similar

1983 ◽  
Vol 213 (2) ◽  
pp. 473-478 ◽  
Author(s):  
J J Hubert ◽  
J S O′Brien
Keyword(s):  
Gel Electrophoresis ◽  
Human Liver ◽  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Major Band ◽  
Chymotryptic Peptide ◽  
Human Acid ◽  
Dog Liver ◽  
Peptide Maps

The purification of dog liver acid β-galactosidase is described. The dog enzyme migrated as a single major band on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, with a molecular weight of 60000. Antiserum raised against purified human liver acid β-galactosidase cross-reacted with β-galactosidase from dog liver, but not with those from cat liver or Escherichia coli. Tryptic peptide maps of the dog and human acid β-galactosidases indicate that 21 of the 24 peptides observed were homologous; a similar result was obtained after chymotryptic peptide mapping. We conclude that dog and human acid β-galactosidases are structurally similar, and that canine GM1 gangliosidosis (acid β-galactosidase deficiency) is an excellent model for the same disease in man.

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