scholarly journals Isolation and characterization of plasma-membrane glycoproteins from pig epidermis

1982 ◽  
Vol 201 (2) ◽  
pp. 287-295 ◽  
Author(s):  
Ian A. King ◽  
Anne Tabiowo
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Plasma Membranes ◽  
Periodic Acid ◽  
Marker Enzymes ◽  
Membrane Glycoproteins ◽  
Periodic Acid Schiff ◽  
Isolation And Characterization ◽  
Lentil Lectin ◽  
Sepharose 4B

1. Non-desmosomal plasma membranes enriched in plasma-membrane marker enzymes and in metabolically labelled glycoproteins were isolated on a large scale from up to 500g of pig ear skin slices. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and periodic acid/Schiff staining revealed the presence of four major glycosylated components in the apparent molecular-weight range 150000–80000. 2. A large proportion of the marker enzymes, the d-[3H]glucosamine-labelled glycoproteins and the periodic acid/Schiff-stained glycoproteins were solubilized by 1% (w/v) sodium deoxycholate. However, several non-glycosylated proteins, in particular those with mol.wts. 81000, 41000 and 38000 (possibly cytoskeletal components), were relatively resistant to solubilization. 3. The deoxycholate-solubilized membranes were fractionated by lectin affinity chromatography using both concanavalin A–Sepharose 4B and lentil lectin–Sepharose 4B. From 75 to 85% of the applied glycoprotein was recovered from the columns. From 30 to 40% of the recovered glycoprotein was specifically bound by the lectins and was eluted with 2% (w/v) α-methyl d-mannoside. The enrichment of labelled glycoproteins in the material bound by the lectins (2.5-fold) was similar with both lectins, although the yield was somewhat greater when lentil lectin was used. The glycoprotein-enriched fraction was also enriched in all the plasma-membrane marker enzymes, indicating their probable glycoprotein nature. 4. The glycoprotein-enriched fraction contained the four major periodic acid/Schiff-stained bands that were detected in the original plasma membrane. They had apparent mol.wts. 147000, 130500, 108000 and 91400. The higher-molecular-weight components contained relatively more d-[3H]glucosamine, indicating differences in the sugar composition or in the metabolic turnover of the individual glycoproteins in culture. The material bound by the lectins also contained a number of lower-molecular-weight Coomassie Brilliant Blue-stained components. These were weakly stained by periodic acid/Schiff reagent and were lightly labelled with d-[3H]glucosamine, indicating that they contained less carbohydrate than the four major glycoprotein bands. 5. Chloroform/methanol-extracted plasma membranes and isolated glycoproteins had a similar carbohydrate composition, containing sialic acid, hexosamine, fucose, xylose, mannose, galactose and glucose. Glucose was not enriched in the isolated glycoproteins, suggesting that it may be a contaminant. Xylose, however, was enriched in the isolated glycoproteins. It remains to be established whether this sugar, which is not usually found in plasma-membrane glycoproteins, is a genuine constituent of plasma-membrane glycoproteins in the epidermis.

scholarly journals Specific protein and glycoprotein deficiencies in platelets isolated from two patients with the gray platelet syndrome

Blood ◽  
1982 ◽  
Vol 59 (4) ◽  
pp. 709-718 ◽  
Author(s):  
AT Nurden ◽  
TJ Kunicki ◽  
D Dupuis ◽  
C Soria ◽  
JP Caen
Keyword(s):  
Molecular Weight ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Blue Staining ◽  
Membrane Glycoproteins ◽  
Periodic Acid Schiff ◽  
Platelet Factor ◽  
Sds Page ◽  
Platelet Disorder ◽  
Gray Platelet Syndrome

Abstract The gray platelet syndrome is a rare inherited platelet disorder characterized by the absence of alp ha-granules as observed by electron microscopy. Analysis of the glycoprotein composition of the platelets of 2 such patients by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed decreased or absent staining for carbohydrate of several high molecular weight glycoproteins. The major periodic acid Schiff (PAS) staining membrane glycoproteins were normally detected and were normally labeled with 125I during lactoperoxidase-catalyzed iodination. Analysis of the protein composition of gray platelets by single or two- dimensional SDS-PAGE followed by Coomassie blue staining revealed an apparent absence of GP Ig (thrombospondin), markedly reduced platelet fibrinogen and albumin concentrations, and severely reduced levels of 2 low molecular weight polypeptides exhibiting identical rates of migration on SDS-PAGE as platelet factor 4 and beta-thromboglobulin. SDS-PAGE profiles similar to those of the gray platelets were observed with normal human platelets that had undergone the release reaction induced by thrombin. Analysis of gray platelet proteins by crossed immunoelectrophoresis using a rabbit anti-human platelet antibody preparation and rocket immunoelectrophoresis using monospecific antisera confirmed the above findings and showed additional severe deficiencies of factor VIIIR:Ag and cold-insoluble globulin. In contrast, factor XIII (subunit A), a cytoplasmic protein, was normally detected. Our studies provide further evidence that circulating gray platelets specifically lack, or have markedly decreased concentrations of, the alpha-granule proteins.

scholarly journals Specific protein and glycoprotein deficiencies in platelets isolated from two patients with the gray platelet syndrome

Blood ◽  
1982 ◽  
Vol 59 (4) ◽  
pp. 709-718 ◽  
Author(s):  
AT Nurden ◽  
TJ Kunicki ◽  
D Dupuis ◽  
C Soria ◽  
JP Caen
Keyword(s):  
Molecular Weight ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Blue Staining ◽  
Membrane Glycoproteins ◽  
Periodic Acid Schiff ◽  
Platelet Factor ◽  
Sds Page ◽  
Platelet Disorder ◽  
Gray Platelet Syndrome

The gray platelet syndrome is a rare inherited platelet disorder characterized by the absence of alp ha-granules as observed by electron microscopy. Analysis of the glycoprotein composition of the platelets of 2 such patients by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed decreased or absent staining for carbohydrate of several high molecular weight glycoproteins. The major periodic acid Schiff (PAS) staining membrane glycoproteins were normally detected and were normally labeled with 125I during lactoperoxidase-catalyzed iodination. Analysis of the protein composition of gray platelets by single or two- dimensional SDS-PAGE followed by Coomassie blue staining revealed an apparent absence of GP Ig (thrombospondin), markedly reduced platelet fibrinogen and albumin concentrations, and severely reduced levels of 2 low molecular weight polypeptides exhibiting identical rates of migration on SDS-PAGE as platelet factor 4 and beta-thromboglobulin. SDS-PAGE profiles similar to those of the gray platelets were observed with normal human platelets that had undergone the release reaction induced by thrombin. Analysis of gray platelet proteins by crossed immunoelectrophoresis using a rabbit anti-human platelet antibody preparation and rocket immunoelectrophoresis using monospecific antisera confirmed the above findings and showed additional severe deficiencies of factor VIIIR:Ag and cold-insoluble globulin. In contrast, factor XIII (subunit A), a cytoplasmic protein, was normally detected. Our studies provide further evidence that circulating gray platelets specifically lack, or have markedly decreased concentrations of, the alpha-granule proteins.

scholarly journals Release of intestinal surface-membrane glycoproteins associated with enzyme activity by brief digestion with papain

1971 ◽  
Vol 121 (5) ◽  
pp. 781-789 ◽  
Author(s):  
Gordon G. Forstner
Keyword(s):  
Brush Border ◽  
Gradient Centrifugation ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Surface Membrane ◽  
Membrane Glycoproteins ◽  
Periodic Acid Schiff ◽  
Brush Borders ◽  
And Function ◽  
Sepharose 4B

Rat intestinal surface-membrane glycoproteins were labelled by intraperitoneal injection of [1-14C]glucosamine 4h before the animals were killed. At this time, density-gradient centrifugation of disrupted brush borders indicated that glycoprotein radioactivity was distributed identically with sucrase, a plasma-membrane marker. Labelled brush borders were digested by papain for brief time-intervals known to release surface-enzyme particles without disruption of the unit membrane. Digestion for 5min released 90% of the surface sucrase, and almost one-half of the brush-border glycoprotein and label. On Sepharose 4B column chromatography most of the glycoprotein and label emerged as a single peak. This peak contained the most actively labelled glycoprotein in the brush border and was closely associated with maltase, sucrase, β-naphthylamidase and alkaline phosphatase. The peak was partially resolved on polyacrylamide-gel electrophoresis into three bands. Each band contained a distinctive enzyme or enzyme pair, and was labelled by [1-14C]glucosamine. No periodic acid–Schiff-negative protein was observed in the peak material. Glycoproteins susceptible to brief digestion with papain are therefore closely linked to released surface-enzyme particles. Intestinal surface glycoproteins are heterogeneous with respect to molecular weight, electrophoretic mobility and function.

Purine nucleoside phosphorylase: Isolation and characterization

1990 ◽  
Vol 55 (12) ◽  
pp. 2987-2999 ◽  
Author(s):  
Katarina Šedivá ◽  
Ivan Votruba ◽  
Antonín Holý ◽  
Ivan Rosenberg
Keyword(s):  
Molecular Weight ◽  
Purine Nucleoside Phosphorylase ◽  
Purine Nucleoside ◽  
Leukemia Cells ◽  
Ph Optimum ◽  
Nucleoside Phosphorylase ◽  
Isolation And Characterization ◽  
Reaction Proceeds ◽  
The Matrix ◽  
Sepharose 4B

Purine nucleoside phosphorylase (PNP) from mouse leukemia cells L1210 was purified to homogeneity by a combination of ion exchange and affinity chromatography using AE-Sepharose 4B and 9-(p-succinylaminobenzyl)hypoxanthine as the matrix and the ligand, respectively. The native enzyme has a molecular weight of 104 000 and consists of three subunits of equal molecular weight of 34 000. The results of isoelectric focusing showed that the enzyme is considerably microheterogeneous over the pI-range 4.0-5.8 and most likely consists of eight isozymes. The temperature and pH-optimum of phosphorolysis, purine nucleoside synthesis and also of transribosylation is identical, namely 55 °C and pH 7.4. The transribosylation reaction proceeds in the presence of phosphate only. The following Km-values (μmol l-1) were determined for phosphorolysis: inosine 40, 2'-deoxyinosine 47, guanosine 27, 2'-deoxyguanosine 32. The Km-values (μmol l-1) of purine riboside and deoxyriboside synthesis are lower than the values for phosphorolysis (hypoxanthine 18 and 34, resp., guanine 8 and 11, resp.). An affinity lower by one order shows PNP for (-D-ribose-1-phosphate, (-D-2-deoxyribose-1-phosphate (Km = 200 μmol l-1 in both cases) and phosphate (Km = 805 μmol l-1). The substrate specificity of the enzyme was also studied: positions N(1), C(2) and C(8) are decisive for the binding of the substrate (purine nucleoside).

scholarly journals Isolation and characterization of rabbit kidney brush borders

1972 ◽  
Vol 128 (5) ◽  
pp. 1319-1328 ◽  
Author(s):  
S. J. Quirk ◽  
G. B. Robinson
Keyword(s):  
Plasma Membranes ◽  
Sucrose Density Gradient ◽  
Molar Ratio ◽  
Rabbit Kidney ◽  
Kidney Cortex ◽  
Marker Enzymes ◽  
Isolation And Characterization ◽  
Adenosine Triphosphatases ◽  
Brush Borders

1. Brush borders were isolated from rabbit kidney-cortex homogenates by rate-zonal centrifugation through a sucrose density gradient in a B-XIV zonal rotor, followed by differential centrifugation. 2. The method of preparation gave brush borders of high purity with a reasonable yield. The morphological appearance supported the evidence from enzymic and chemical investigations, that the brush borders were only slightly contaminated with endoplasmic reticulum, mitochondria, lysosomes and nuclei. 3. The molar ratio of cholesterol to phospholipid lay within the range found in other plasma membranes, but the carbohydrate content was double that found in liver plasma membranes. 4. Alkaline phosphatase, maltase, trehalase and aminopeptidase were major enzymic constituents of the brush borders, and had an approximately equal yield and enrichment, but none of these enzymes fulfilled the criteria for marker enzymes. 5. Mg2+-dependent and Na+,K+-dependent adenosine triphosphatases, although found in brush borders, had low yields and low enrichments.

scholarly journals Purification, characterization and radioimmunoassay of adenosine deaminase from human leukaemic granulocytes

1981 ◽  
Vol 195 (2) ◽  
pp. 389-397 ◽  
Author(s):  
D A Wiginton ◽  
M S Coleman ◽  
J J Hutton
Keyword(s):  
Molecular Weight ◽  
Adenosine Deaminase ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Periodic Acid Schiff ◽  
Apparent Homogeneity

Adenosine deaminase was purified 3038-fold to apparent homogeneity from human leukaemic granulocytes by adenosine affinity chromatography. The purified enzyme has a specific activity of 486 mumol/min per mg of protein at 35 degrees C. It exhibits a single band when subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, non-denaturing polyacrylamide-gel electrophoresis and isoelectric focusing. The pI is 4.4. The enzyme is a monomeric protein of molecular weight 44000. Both electrophoretic behaviour and molecular weight differ from those of the low-molecular-weight adenosine deaminase purified from human erythrocytes. Its amino acid composition is reported. Tests with periodic acid-Schiff reagent for associated carbohydrate are negative. Of the large group of physiological compounds tested as potential effectors, none has a significant effect. The enzyme is specific for adenosine and deoxyadenosine, with Km values of 48 microM and 34 microM respectively. There are no significant differences in enzyme function on the two substrates. erythro-9-(2-Hydroxy non-3-yl) adenine is a competitive inhibitor, with Ki 15 nM. Deoxycoformycin inhibits deamination of both adenosine and deoxyadenosine, with an apparent Ki of 60-90 pM. A specific antibody was developed against the purified enzyme, and a sensitive radioimmunoassay for adenosine deaminase protein is described.

Trypsin-uncoupled synthesis and secretion of yeast invertase: implications for the mechanism of secretion

1979 ◽  
Vol 25 (4) ◽  
pp. 528-534 ◽  
Author(s):  
Bruce E. Holbein ◽  
Denis K. Kidby
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Subcellular Distribution ◽  
Plasma Membranes ◽  
External Surface ◽  
Internal Surface ◽  
Low Molecular Weight ◽  
Synthetase Activity ◽  
Trypsin Treatment ◽  
Yeast Invertase

The subcellular distribution of invertase was examined after synthesis and secretion by sphaeroplasts had been uncoupled by the addition of 30 μg mL−1 trypsin. Sphaeroplasts secreted only the high molecular weight invertase during uncoupling by trypsin. The level of low molecular weight, 'small' invertase in the soluble internal pool was found to be elevated by over fivefold, and the membrane-associated pool was found to contain low molecular weight invertase in addition to intermediate molecular weight invertase, after 1.5 h of trypsin treatment. Purified plasma membranes from trypsin-treated sphaeroplasts had no detectable mannan synthetase activity. On the basis of these and previous findings, a working hypothesis wherein invertase is synthesized on the internal surface of the plasma membrane and glycosylated during its transit to the external surface is presented.

scholarly journals Partial purification and characterization of oestrogen receptors in subfractions of hepatocyte plasma membranes

1980 ◽  
Vol 191 (3) ◽  
pp. 743-760 ◽  
Author(s):  
Richard J. Pietras ◽  
Clara M. Szego
Keyword(s):  
Plasma Membrane ◽  
Binding Sites ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Low Density ◽  
Marker Enzymes ◽  
Low Density Plasma ◽  
Density Plasma

To assess the subcellular distribution of oestrogen-binding components in their native state, plasma membrane and other cell fractions were prepared from hepatocytes in the absence of [3H]oestradiol-17β. Cells from livers of ovariectomized rats were disrupted, with submaximal homogenization in buffered isotonic sucrose with CaCl2 and proteinase inhibitor, and fractionated by using isotonic media. Fractions were characterized by determinations of enzyme activities, biochemical constituents and ligand binding. Specific binding of 2nm-[3H]oestradiol-17β to intact cells and their fractions was detemined after equilibration for 1.5h at 4°C. More than 92% of the radioactivity from representative preparations was verified as authentic oestradiol by thin-layer chromatography. Activities of plasma-membrane marker enzymes as well as binding sites for oestrogen and for wheat germ agglutinin were present principally in particulate fractions, rather than in 105000g-supernatant fractions. However, by using alternative homogenization procedures (i.e. hypotonic media), known to fragment and strip structural components, oestradiol-binding sites and activities of plasma-membrane marker enzymes were distributed predominantly into cytosol. By using the more conservative procedures, plasma membranes of low (ρ=1.13–1.16) and high (ρ=1.16–1.18) density were purified from crude nuclear fractions. A second low-density subfraction of plasma membrane was prepared from microsome-rich fractions. Activities of plasma-membrane marker enzymes were enriched to about 28 and four times that of the homogenate in plasma membranes of low and high density respectively. Binding sites for wheat germ agglutinin and oestradiol were concentrated in low-density plasma membranes to 46–63 times that of the homogenate. Specific binding of oestrogen in low-density plasma membranes purified from crude nuclei was saturable, with an apparent association constant of 3.5nm. At saturation, such oestradiol receptors corresponded to 526fmol/mg of membrane protein. A Hill plot showed a moderate degree of positive co-operativity in the interaction of hormone with plasma membranes. Specific binding of [3H]oestradiol-17β was reduced by a 200-fold molar excess of unlabelled oestradiol-17β, oestriol or diethylstilbestrol, but not by oestradiol-17α, cortisol, testosterone or progesterone. Binding was also blocked by prior exposure of membranes to trypsin or to 60°C, but remained essentially undiminished by extraction of membranes with either hypotonic or high-salt buffers. Extraction with 0.1% (v/v) Triton X-100 partially solubilized the oestrogen-binding component(s) of plasma membranes. Particle-free extracts were resolved on 5–20% (w/v) sucrose density gradients with either 0.01m- or 0.4m-KCl, and the fractions were analysed by adsorption to hydroxyapatite. In low-salt gradients macromolecule-bound oestrogen sedimented at predominantly 7.4S and binding was 1560 times that of the homogenate. Under high-salt conditions oestradiol-binding activity occurred at both 3.6S and 4.9S.

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