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.

Origin of periodic acid-Schiff-reactive glycoprotein in human eccrine sweat

1986 ◽  
Vol 60 (5) ◽  
pp. 1615-1622 ◽  
Author(s):  
S. Yanagawa ◽  
H. Yokozeki ◽  
K. Sato
Keyword(s):  
Serum Proteins ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecylsulfate ◽  
Electrophoretic Pattern ◽  
Periodic Acid Schiff ◽  
Dark Cell ◽  
Dark Cells ◽  
Sds Page ◽  
Positive Materials

To evaluate the possible involvement of ductal blockade with periodic acid-Schiff (PAS)-positive materials in the mechanism of hidromeiosis in humans, skin slices were incubated with methacholine for 2 h and PAS-positive materials localized histologically in the ductal lumen. In 20% of the glands complete ductal blockade with PAS-positive materials was noted. The characteristics and origin of such PAS-positive glycoproteins in human sweat were then studied using various electrophoretic techniques. One-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis (1-D SDS-PAGE) demonstrated considerable individual variation in the electrophoretic pattern; however, four major bands at 45, 28, 20, and 18K shared by different individuals, were PAS positive. Further studies using two-dimensional SDS-PAGE, immunodiffusion and immunoaffinity chromatography demonstrated that the PAS-positive glycoproteins are not derived directly from serum because they are electrophoretically and antigenically distinct from serum proteins, including alpha 1-glycoprotein, alpha 2-HS-glycoprotein, and alpha 1-antitrypsin. Since only dark cell granules are densely stained in the histochemical PAS staining, and because antiserum produced against the PAS-positive band selectively stained cells facing the secretory coil lumen (which are most likely dark cells), it is suggested that PAS-positive sweat glycoproteins are derived predominantly from the dark cells.

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.

scholarly journals Detection in milk of a 60,000 Mr mouse and a 68,000 Mr human phosphorylated glycoprotein by histochemical analysis on polyacrylamide gels.

1983 ◽  
Vol 31 (6) ◽  
pp. 709-716 ◽  
Author(s):  
M R Green
Keyword(s):  
Molecular Weight ◽  
Sialic Acid ◽  
Periodic Acid ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Blue Staining ◽  
Polyacrylamide Gels ◽  
Periodic Acid Schiff ◽  
Coomassie Blue Staining ◽  
Coomassie Blue

Proteins in colostrum and skimmed milk from humans and mice were separated by electrophoresis on polyacrylamide gels and stained with Coomassie blue (CB), Ethyl-Stains-all (ESA), and periodic acid-Schiff (PAS) to investigate changes that may occur in milks throughout lactation. In mouse colostrum but not in mature mouse milk, a PAS-positive protein of apparent molecular weight of 60,000 stained prominently blue with ESA. A protein in human milk with a molecular weight of 68,000 stained similarly but was present throughout lactation. The intensity of blue staining of these minor proteins in milk approached that obtained with casein phosphoproteins. The metachromatic dye ESA stains phosphoproteins and sialic acid-rich glycoproteins blue to blue-green. Removal of phosphorus from the former and sialic acid from the latter results in those proteins staining red with ESA. The intensity of blue staining of the 60,000 and 68,000 Mr proteins was diminished but not lost following treatment with phosphatase. It was eliminated following neuraminidase digestion of the mouse protein and mild acid hydrolysis of the human protein. Coomassie blue staining of the proteins was not affected by these procedures. Following electrophoresis of milk and milk fractions in a non-sodium dodecyl sulfate-containing system, the proteins were identified by their characteristic staining properties with ESA and isolated.

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 Human platelet-specific antigen, Siba, is associated with the molecular weight polymorphism of glycoprotein Ib alpha

Blood ◽  
1991 ◽  
Vol 78 (7) ◽  
pp. 1722-1729 ◽  
Author(s):  
F Ishida ◽  
H Saji ◽  
E Maruya ◽  
K Furihata
Keyword(s):  
Molecular Weight ◽  
Human Platelet ◽  
Close Association ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Specific Antigen ◽  
Human Leukocyte ◽  
Leukocyte Antigen ◽  
Antigen Capture ◽  
Sds Page

Abstract Platelet-specific antigen Sib(a) has been highly implicated in the pathogenesis of refractoriness to human leukocyte antigen (HLA)-matched platelet transfusions in Japan. We provide evidence that the Sib(a) antigen is located on the glycoprotein (GP) Ib alpha and has a close association with the molecular weight (mol wt) polymorphism of GPIb. In modified antigen-capture ELISA (MACE), anti-Sib(a) antibody reacted only with GPIb/IX held by a murine anti-GPIb/IX monoclonal antibody (MoAb). The reactivity of anti-Sib(a) antibody to Sib(a)-positive (Sib(a+)) platelets was abolished after they were treated with Serratia marcescens protease. Platelets from 50 healthy volunteers were semiquantitatively phenotyped for Sib(a) antigen by MACE and divided into three distinct groups: strongly positive, positive, and negative. They were also analyzed by sodium dodeyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and periodic acid-silver staining for mol wt polymorphism of GPIb, phenotyped as A, B, C, or D. Without exception, Sib(a+) platelets showed larger phenotypes (A or B). Removal of sialic acid from Sib(a+) platelets did not reduce the binding of anti-Sib(a). Finally, anti-Sib(a) antibody specifically immunoprecipitated A and B phenotypes of GPIb from Sib(a+) platelets. Thus, Sib(a) antigen evidently is located in the region of glycocalicin that is present only on the A and B phenotypes of GPIb.

Tryptic Peptide Maps Of The Major Human Blood Platelet Membrane Glycoproteins, Ia, IIa, IIb, IIIa, IIIb and IIIc

1981 ◽  
Author(s):  
J L McGregor ◽  
K J Clemetson ◽  
E James ◽  
P Clezardin ◽  
M Dechavanne ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Tryptic Peptide ◽  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Platelet Membrane ◽  
Blue Staining ◽  
Membrane Glycoproteins ◽  
Lectin Affinity Chromatography ◽  
Reducing Conditions

Some major platelet membrane glycoproteins separated by 2-dimensional polyacrylamide gel electrophoresis (isoelectric focusing, discontinuous SDS-gel electrophoresis) have been characterized by 2-dimensional tryptic peptide mapping. Human platelets were isolated, washed and surface-labelled by lactoperoxidase-catalyzed iodination. Labelled platelets were solubilized in SDS and separated by 2-dimensional gel electrophoresis under non-reducing conditions in the 1st dimension and either reducing or non-reducing conditions in the 2nd dimension. Alternatively, labelled platelets were solubilized in sodium deoxy- cholate (1 %) and the glycosylated components were isolated by lectin affinity-chromatography on Lens culinaris lectin or concanavalin A lectin and separated by 2-dimensional gel electrophoresis. The glycoproteins were cut out from 6 % polyacrylamide gels, after being identified with Coomassie blue staining and indirect autoradiography by their pI and molecular weight. Tryptic maps were prepared according to the method of Elder et al. The tryptic maps of GPIa, IIa, IIb, IIIa, IIIb and IIIc are different, with each showing a characteristic pattern with the possible exception of Ia and IIb which showed certain similarities in both the reduced and non-reduced states. GPIIa which is clearly separated under non-reducing conditions, appears from its tryptic map to be present in the Ib region when reduced. Thus this technique clearly identifies each GP by a parameter in addition to pI and molecular weight.

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.

scholarly journals Human platelet-specific antigen, Siba, is associated with the molecular weight polymorphism of glycoprotein Ib alpha

Blood ◽  
1991 ◽  
Vol 78 (7) ◽  
pp. 1722-1729 ◽  
Author(s):  
F Ishida ◽  
H Saji ◽  
E Maruya ◽  
K Furihata
Keyword(s):  
Molecular Weight ◽  
Human Platelet ◽  
Close Association ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Specific Antigen ◽  
Human Leukocyte ◽  
Leukocyte Antigen ◽  
Antigen Capture ◽  
Sds Page

Platelet-specific antigen Sib(a) has been highly implicated in the pathogenesis of refractoriness to human leukocyte antigen (HLA)-matched platelet transfusions in Japan. We provide evidence that the Sib(a) antigen is located on the glycoprotein (GP) Ib alpha and has a close association with the molecular weight (mol wt) polymorphism of GPIb. In modified antigen-capture ELISA (MACE), anti-Sib(a) antibody reacted only with GPIb/IX held by a murine anti-GPIb/IX monoclonal antibody (MoAb). The reactivity of anti-Sib(a) antibody to Sib(a)-positive (Sib(a+)) platelets was abolished after they were treated with Serratia marcescens protease. Platelets from 50 healthy volunteers were semiquantitatively phenotyped for Sib(a) antigen by MACE and divided into three distinct groups: strongly positive, positive, and negative. They were also analyzed by sodium dodeyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and periodic acid-silver staining for mol wt polymorphism of GPIb, phenotyped as A, B, C, or D. Without exception, Sib(a+) platelets showed larger phenotypes (A or B). Removal of sialic acid from Sib(a+) platelets did not reduce the binding of anti-Sib(a). Finally, anti-Sib(a) antibody specifically immunoprecipitated A and B phenotypes of GPIb from Sib(a+) platelets. Thus, Sib(a) antigen evidently is located in the region of glycocalicin that is present only on the A and B phenotypes of GPIb.

Heterozygous Abnormal Fibrinogen Osaka III with the Replacement of γ Arginine-275 by Histidine Has an Apparently Higher Molecular Weight γ-Chain Variant

1992 ◽  
Vol 68 (05) ◽  
pp. 534-538 ◽  
Author(s):  
Nobuhiko Yoshida ◽  
Shingi Imaoka ◽  
Hajime Hirata ◽  
Michio Matsuda ◽  
Shinji Asakura
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tetraacetic Acid ◽  
Fibrin Monomer ◽  
Sds Page ◽  
Thrombotic Tendency ◽  
Chain Variant

SummaryCongenitally abnormal fibrinogen Osaka III with the replacement of γ Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of α- and γ-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal γ-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 γ remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethy-lenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

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