DirectN-terminal sequence analysis of rat liver plasma membrane glycoproteins separated by two-dimensional polyacrylamide gel electrophoresis

Two membrane glycoproteins that bound immune complexes and inhibited Fc-receptor- (FcR-)mediated functions in vitro were purified from human FcR+ chronic-lymphocytic-leukaemia cells. A multi-step purification was developed, consisting essentially in: (i) Tween 40 extraction of crude cell membranes; (ii) solubilization of membrane fragments by Renex-30; (iii) isolation of glycoproteins by affinity chromatography on Lens culinaris haemagglutinin-Sepharose; (iv) papain treatment of the eluted glycoproteins followed by gel-filtration chromatography; (v) purification by polyacrylamide-gel electrophoresis of two molecular species from the protein-size fraction enriched for immune-complex-binding activity. The two electrophoretically isolated components displayed apparent molecular masses of 70 and 45 kDa by SDS/polyacrylamide-gel electrophoresis and restricted charge heterogeneity by two-dimensional analysis. Two-dimensional peptide mapping revealed the presence of many peptides in common between the two proteins and the absence of a number of peptides in the 45 kDa component. These two polypeptides were used as immunogens to produce polyclonal antibodies that cross-reacted with both proteins and specifically inhibited FcR-mediated reactions in vitro. Furthermore, FcR-related components from detergent-extracted lysates of the human K562 and U937 cell lines or human placental membranes were revealed by the putative anti-FcR antibodies adsorbed on Protein A-Sepharose.

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Studies on proteins of animal ribosomes. XIV. analysis of phosphorylated rat liver ribosomal proteins by two-dimensional polyacrylamide gel electrophoresis

FEBS Letters ◽

10.1016/0014-5793(72)80580-5 ◽

1972 ◽

Vol 26 (1-2) ◽

pp. 233-236 ◽

Cited By ~ 23

Author(s):

J. Stahl ◽

H. Welfle ◽

H. Bielka

Keyword(s):

Rat Liver ◽

Gel Electrophoresis ◽

Ribosomal Proteins ◽

Polyacrylamide Gel Electrophoresis ◽

Polyacrylamide Gel ◽

Two Dimensional

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Additional glycoprotein defects in Bernard-Soulier's syndrome: confirmation of genetic basis by parental analysis

Blood ◽

10.1182/blood.v62.4.800.800 ◽

1983 ◽

Vol 62 (4) ◽

pp. 800-807 ◽

Cited By ~ 120

Author(s):

MC Berndt ◽

C Gregory ◽

BH Chong ◽

H Zola ◽

PA Castaldi

Keyword(s):

Molecular Weight ◽

Gel Electrophoresis ◽

Membrane Surface ◽

Polyacrylamide Gel Electrophoresis ◽

Polyacrylamide Gel ◽

Two Dimensional ◽

Membrane Glycoproteins ◽

Glycoprotein Ib ◽

Membrane Complex ◽

Platelet Protein

Abstract The glycoprotein profile of Bernard-Soulier platelets was examined by labeling washed platelets with periodate 3H-sodium borohydride, a procedure that labels greater than 30 glycoproteins on the membrane surface of normal platelets. Three Bernard-Soulier patients were studied; two were siblings and the third was unrelated. The platelet protein and glycoprotein profiles were evaluated under nonreduced and reduced conditions using 5%-15% exponential SDS-polyacrylamide gel electrophoresis. The two siblings completely lacked glycoprotein Ib (GPIb). The unrelated patient had congruent to 7% of the normal level. This was confirmed by two-dimensional nonreduced-reduced SDS- polyacrylamide gel electrophoresis, a procedure that allows clear separation of the disulfide-linked subunits of GPIb, GPIb alpha (mol wt 145,000), and GPIb beta (mol wt 25,000) from other membrane glycoproteins. On one-dimensional analysis, Bernard-Soulier's syndrome (BSS) platelets also lacked the peripheral membrane glycoprotein, GPV (mol wt 82,000) and a low molecular weight glycoprotein, GPIX, (nonreduced or reduced, mol wt congruent to 22,000). The two- dimensional gel system also revealed the absence of a minor glycoprotein with a molecular weight of congruent to 100,000 (GP 100). Quantitation of these proteins solubilized from electrophoretograms showed that the siblings' parents had congruent to 50% levels of GPIb, GPIX, and GP 100. A monoclonal antibody against glycoprotein Ib, FMC 25, was negative by immunofluorescence against Bernard-Soulier platelets and immuneprecipitated both GP Ib and GPIX from Triton X100 solubilized, labeled platelets. The combined results suggest that the apparent genetic absence of multiple proteins in Bernard-Soulier platelets is due, in part, to the presence in normal platelets of a tight membrane complex between glycoprotein Ib and at least one of the other absent glycoproteins.

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Effect of tunicamycin on epidermal glycoprotein and glycosaminoglycan synthesis in vitro

Biochemical Journal ◽

10.1042/bj1980331 ◽

1981 ◽

Vol 198 (2) ◽

pp. 331-338 ◽

Cited By ~ 26

Author(s):

Ian A. King ◽

Anne Tabiowo

Keyword(s):

Amino Acids ◽

Plasma Membrane ◽

Cell Adhesion ◽

Epidermal Cell ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Polyacrylamide Gel ◽

Membrane Glycoproteins ◽

Glycosaminoglycan Synthesis ◽

Core Proteins

1. When pig ear skin slices were cultured for 18h in the presence of 1mug of tunicamycin/ml the incorporation of d-[3H]glucosamine into the epidermis, solubilized with 8m-urea/5% (w/v) sodium dodecyl sulphate, was inhibited by 45–55%. This degree of inhibition was not increased by using up to 5mug of tunicamycin/ml or by treating the skin slices with tunicamycin for up to 8 days. The incorporation of (U-14C)-labelled l-amino acids under these conditions was not affected by tunicamycin. Polyacrylamide-gel electrophoresis indicated that the labelling of the major glycosaminoglycan peak with d-[3H]glucosamine was unaffected, whereas that of the faster migrating glycoprotein components was considerably decreased in the presence of tunicamycin. 2. Subcellular fractionation indicated that tunicamycin specifically inhibited the incorporation of d-[3H]glucosamine but not of (U-14C)-labelled l-amino acids into particulate (mainly plasma-membrane) glycoproteins by about 70%. The labelling of soluble glycoproteins was hardly affected. Polyacrylamide-gel electrophoresis of the plasma-membrane fraction showed decreased d-[3H]glucosamine incorporation into all glycoprotein components, indicating that the plasma-membrane glycoproteins contained mainly N-asparagine-linked oligosaccharides. 3. Cellulose acetate electrophoresis of both cellular and extracellular glycosaminoglycans showed that tunicamycin had no significant effect on the synthesis of the major component, hyaluronic acid. However, the incorporation of both d-[3H]glucosamine and 35SO42− into sulphated glycosaminoglycans was inhibited by about 50%. This inhibition was partially overcome, at least in the cellular fraction, by 2mm-p-nitrophenyl β-d-xyloside indicating that tunicamycin-treated epidermis retained the ability to synthesize sulphated glycosaminoglycan chains. Tunicamycin may affect the synthesis and/or degradation of proteoglycan core proteins or the xylosyltransferase. 4. Electron-microscopic examination of epidermis treated with tunicamycin for up to 4 days revealed no significant changes in cell-surface morphology or in epidermal-cell adhesion. Either N-asparagine-linked carbohydrates play little role in epidermal-cell adhesion or more probably there is little turnover of these components in epidermal adhesive structures such as desmosomes and hemidesmosomes during organ culture.

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Additional glycoprotein defects in Bernard-Soulier's syndrome: confirmation of genetic basis by parental analysis

Blood ◽

10.1182/blood.v62.4.800.bloodjournal624800 ◽

1983 ◽

Vol 62 (4) ◽

pp. 800-807 ◽

Cited By ~ 2

Author(s):

MC Berndt ◽

C Gregory ◽

BH Chong ◽

H Zola ◽

PA Castaldi

Keyword(s):

Molecular Weight ◽

Gel Electrophoresis ◽

Membrane Surface ◽

Polyacrylamide Gel Electrophoresis ◽

Polyacrylamide Gel ◽

Two Dimensional ◽

Membrane Glycoproteins ◽

Glycoprotein Ib ◽

Membrane Complex ◽

Platelet Protein

The glycoprotein profile of Bernard-Soulier platelets was examined by labeling washed platelets with periodate 3H-sodium borohydride, a procedure that labels greater than 30 glycoproteins on the membrane surface of normal platelets. Three Bernard-Soulier patients were studied; two were siblings and the third was unrelated. The platelet protein and glycoprotein profiles were evaluated under nonreduced and reduced conditions using 5%-15% exponential SDS-polyacrylamide gel electrophoresis. The two siblings completely lacked glycoprotein Ib (GPIb). The unrelated patient had congruent to 7% of the normal level. This was confirmed by two-dimensional nonreduced-reduced SDS- polyacrylamide gel electrophoresis, a procedure that allows clear separation of the disulfide-linked subunits of GPIb, GPIb alpha (mol wt 145,000), and GPIb beta (mol wt 25,000) from other membrane glycoproteins. On one-dimensional analysis, Bernard-Soulier's syndrome (BSS) platelets also lacked the peripheral membrane glycoprotein, GPV (mol wt 82,000) and a low molecular weight glycoprotein, GPIX, (nonreduced or reduced, mol wt congruent to 22,000). The two- dimensional gel system also revealed the absence of a minor glycoprotein with a molecular weight of congruent to 100,000 (GP 100). Quantitation of these proteins solubilized from electrophoretograms showed that the siblings' parents had congruent to 50% levels of GPIb, GPIX, and GP 100. A monoclonal antibody against glycoprotein Ib, FMC 25, was negative by immunofluorescence against Bernard-Soulier platelets and immuneprecipitated both GP Ib and GPIX from Triton X100 solubilized, labeled platelets. The combined results suggest that the apparent genetic absence of multiple proteins in Bernard-Soulier platelets is due, in part, to the presence in normal platelets of a tight membrane complex between glycoprotein Ib and at least one of the other absent glycoproteins.

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