Organization of Glycoprotein and Glycolipid in the Plasma Membrane of Normal and Transformed Cells as Revealed by Galactose Oxidase

Biomembranes ◽  
1976 ◽  
pp. 131-165 ◽  
Author(s):  
Carl G. Gahmberg ◽  
Sen-itiroh Hakomori
Keyword(s):  
Plasma Membrane ◽  
Galactose Oxidase ◽  
Transformed Cells

scholarly journals Surface glycoproteins of human spermatozoa

1986 ◽  
Vol 82 (1) ◽  
pp. 11-22
Author(s):  
M. Kallajoki ◽  
I. Virtanen ◽  
J. Suominen
Keyword(s):  
Plasma Membrane ◽  
Gel Electrophoresis ◽  
Soluble Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Human Spermatozoa ◽  
Galactose Oxidase ◽  
Sperm Cells ◽  
Neuraminidase Treatment ◽  
Triton X

The surface membrane glycoprotein composition of human spermatozoa has been studied by introducing radioactive label into galactosyl (Gal) and N-acetylgalactosaminyl (GalNAc) residues by using the galactose oxidase/NaB3H4 method. Triton X-100 extracts and Triton X-100-resistant cytoskeletal residues were subjected to analysis by polyacrylamide gel electrophoresis. The distribution of the radiolabel in sperm cells was studied by light-microscopic auto-radiography. The grains were evenly distributed on the cells by the labelling methods used. The Triton X-100 treatment did not affect sperm morphology at the light-microscopic level, but in transmission electron microscopy the plasma membrane covering the acrosome was removed totally, together with most of the acrosomal membranes and acrosomal contents. Plasma membrane residues were, however, always found in the postacrosomal region. Borohydride alone without oxidative pretreatment labelled two polypeptides of molecular weights (Mr) 48,000 and 43,000 in the Triton X-100-soluble fraction. When the Gal/GalNAc residues were labelled by galactose oxidase pretreatment 120,000, 105,000, 78,000 and 68,000 Mr glycoproteins were revealed. When additional neuraminidase treatment was used to remove terminal sialic acid residues, the total labelling intensity was increased two- to fivefold and additional 36,000 and 20,000 Mr glycoproteins were revealed. The Triton X-100-resistant cytoskeletal residue contained 53–75% of the total radioactivity bound in sperm cells. When these components were analysed by polyacrylamide gel electrophoresis, all the major bands found in the Triton X-100-soluble fraction were detected and also some radioactivity was incorporated into the major bands visualized by protein staining. In the present study we describe several human sperm glycoproteins, which seem to be distributed evenly on the sperm cells. Detergent extraction, producing cytoskeletal models, appeared to leave most of the glycoproteins detectable in the extraction residues also with the apparent enrichment of a single 68,000 Mr glycoprotein.

Cytochalasin B induces polarisation of plasma membrane components and actin in transformed cells

Nature ◽  
1978 ◽  
Vol 274 (5674) ◽  
pp. 915-917 ◽  
Author(s):  
K.-G. SUNDQVIST ◽  
P. OTTESKOG ◽  
T. EGE
Keyword(s):  
Plasma Membrane ◽  
Cytochalasin B ◽  
Transformed Cells ◽  
Membrane Components

Macromolecular Glucosamine-Containing Component of the Surface of Cultivated Mouse Cells

1970 ◽  
Vol 7 (2) ◽  
pp. 337-355
Author(s):  
K. ONODERA ◽  
ROSE SHEININ
Keyword(s):  
Plasma Membrane ◽  
Cell Division ◽  
Cell Surface ◽  
Plating Efficiency ◽  
Transformed Cells ◽  
3T3 Cells ◽  
Vital Stain ◽  
Surface Component ◽  
Mouse Cells ◽  
Macromolecular Component

It has been demonstrated that a glucosamine-containing macromolecular component of the cell surface of 3T3 mouse cells, and SV40-transformed cells, is released from cells by treatment with trypsin under conditions in which the plasma membrane remains functionally intact. This was shown by the fact that the treated cells could be cloned with high plating efficiency and remained impermeable to the vital stain, erythrocin. A method for specifically marking this surface component has been devised based on the finding that in 3T3 cells growing synchronously after subculture by trypsin maximum incorporation of glucosamine into this material occurs 12-13 h thereafter. Of the total radioactive glucosamine incorporated into macro-molecular cell constituents, over 80% was recovered in surface component. Studies on the biosynthesis of surface component revealed that this was periodic during a cycle of cell duplication, with an increased rate of formation immediately after cell division. It was found that the surface component of 3T3 cells differed from that of SV40-transformed cells.

Smooth muscle membrane vesicle orientation: a study on intactness and sidedness of rat myometrium plasma membrane vesicles

1980 ◽  
Vol 58 (10) ◽  
pp. 1202-1211 ◽  
Author(s):  
A. K. Grover ◽  
J. Crankshaw ◽  
R. E. Garfield ◽  
E. E. Daniel
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Shock Treatment ◽  
Galactose Oxidase ◽  
Muscle Membrane ◽  
Plasma Membrane Vesicles ◽  
Binding Studies ◽  
Hypotonic Shock

Plasma membrane vesicles of rat myometrium were prepared in media containing 240 mM sucrose. The vesicles were exposed to isotonic, hypertonic, and hypotonic sucrose concentrations, fixed, sectioned, and studied using the electron microscope. The vesicles fixed in isotonic media were circular in appearance. Vesicles fixed in hypertonic media were distorted and showed a reduced volume to surface ratio consistent with the hypothesis that >80% of the vesicles were osmotically active to sucrose. Cationized ferritin binding studies and Ca binding and release studies were also consistent with this finding. Exposure to hypotonic media also yielded membranes with distorted profiles indicating that they had been ruptured. [3H]Sucrose trapping experiments revealed that the vesicles had an internal volume of 1.20–1.44 mL/g protein. Hypotonic shock treatment reduced this intravesicular volume to 0.20–0.28 mL/g protein. The hypotonic shock treatment also led to enhanced galactose oxidase catalyzed Na3B3H4 labelling of the membranes and to increased K+-activated ouabain-sensitive p-nitrophenyl phosphatase activity. The enhancement was the same (55 ± 10%) in the various membrane preparations for both the parameters. The data are interpreted to conclude that the rat myometrium plasma membrane vesicles consisted of 20% broken vesicles and equal proportions of intact vesicles of inside-out and rightside-out orientations.

scholarly journals Neutrophil plasma membranes. I. High-yield purification of human neutrophil plasma membrane vesicles by nitrogen cavitation and differential centrifugation.

1980 ◽  
Vol 86 (1) ◽  
pp. 21-28 ◽  
Author(s):  
M S Klempner ◽  
R B Mikkelsen ◽  
D H Corfman ◽  
J André-Schwartz
Keyword(s):  
Plasma Membrane ◽  
Human Neutrophil ◽  
Plasma Membranes ◽  
Hydrolytic Enzymes ◽  
Membrane Vesicles ◽  
Galactose Oxidase ◽  
High Yield ◽  
Differential Centrifugation ◽  
Plasma Membrane Vesicles ◽  
Equilibrium Ultracentrifugation

Neutrophil chemotaxis, phagocytosis, and oxygen-dependent microbicidal activity are initiated by interactions of stimuli with the plasma membrane. However, difficulties in neutrophil plasma membrane isolation have precluded studies on the precise structure or function of this cellular component. In this paper, a method is described for the isolation of representative human neutrophil plasma membrane vesicles, using nitrogen cavitation for cell disruption and a combination of differential centrifugation and equilibrium ultracentrifugation in Dextran gradients for membrane fractionation. Multiple biochemical markers and galactose oxidase-tritiated sodium borohydride surface labeling were employed to follow the yield, purity, and distribution of plasma membranes, nuclei, lysosomes, endoplasmic reticulum, mitochondria, and cytosol. According to these markers, neutrophil plasma membranes were exposed to minimal lysosomal hydrolytic enzymes and could be isolated free of other subcellular organelles. In contrast, disruption of neutrophils by mechanical homogenization resulted in > 20% lysosomal rupture and significant plasma membrane proteolysis. Electron microscopy demonstrated that plasma membranes isolated after nitrogen cavitation appeared to be sealed vesicles with striking homogeneity.

Characterization and partial purification of a plasma membrane receptor for Bacillus thuringiensis var. kurstaki lepidopteran-specific delta-endotoxin

1986 ◽  
Vol 83 (1) ◽  
pp. 89-101
Author(s):  
B.H. Knowles ◽  
D.J. Ellar
Keyword(s):  
Plasma Membrane ◽  
Bacillus Thuringiensis ◽  
Choristoneura Fumiferana ◽  
Galactose Oxidase ◽  
Partial Purification ◽  
Wide Range ◽  
Delta Endotoxin ◽  
Plasma Membrane Receptor ◽  
Insect Gut

The lepidopteran-specific P1 delta-endotoxin of Bacillus thuringiensis var. kurstaki HD-1 was activated in vitro using insect gut proteases and found to be highly specific for the lepidopteran cell line Choristoneura fumiferana CF1 among a wide range of lepidopteran and dipteran cell lines tested. The toxicity of P1 against CF1 cells is inhibited by N-acetylgalactosamine (GalNAc), and the lectins soybean agglutinin (SBA) and wheat-germ agglutinin. Protein blotting was used to identify a glycoprotein of 146 X 10(3) Mr in the plasma membrane of CF1 cells, capable of binding both the toxin and SBA, which is specific for GalNAc. This glycoprotein was labelled using galactose oxidase and sodium boro-[3H]hydride and solubilized in Triton X-100 before partial purification by affinity chromatography on SBA-agarose. We propose that this glycoprotein is a good candidate for the cellular receptor of the lepidopteran-specific P1 delta-endotoxin of B. thuringiensis var. kurstaki HD-1.

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