scholarly journals Two-dimensional polyacrylamide-gel electrophoresis of the proteins and glycoproteins of purified human platelet surface and intracellular membranes

1984 ◽  
Vol 222 (1) ◽  
pp. 235-246 ◽  
Author(s):  
N Hack ◽  
N Crawford
Keyword(s):  
Concanavalin A ◽  
Gel Electrophoresis ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Surface Membrane ◽  
Actin Binding ◽  
Good Resolution ◽  
Intracellular Membrane ◽  
Neuraminidase Treatment ◽  
Platelet Surface

By using highly purified surface and intracellular membrane fractions prepared from human platelets by free-flow electrophoresis, the polypeptide and glycopeptides of these membranes have been characterized by high-resolution gel electrophoresis under reducing and non-reducing conditions. Silver staining and a variety of glycoprotein-staining procedures have been applied to identify the major components. The principal finding was the clear disparity between the distribution patterns for these two membrane fractions. There are proportionately more low-Mr acidic components present in the intracellular membrane than in the surface-derived membrane. Of the major platelet surface glycoproteins GPIb, IIb, IIIa and IIIb (or IV) well expressed in the surface membrane only, GPIIb and IIIa appear as trace components in the intracellular membrane. The cytoskeleton proteins, actin, myosin, tropomyosin, actin-binding protein and alpha-actinin are prominent features of the surface membrane and essentially absent from the intracellular membrane. Neuraminidase treatment at the whole-cell level, before homogenization, which is an essential requirement for good resolution of the two membrane subfractions, modifies a number of the glycoprotein subunits with respect to their pI characteristics, suggesting much molecular micro-heterogeneity with respect to sialic acid content. A comparison of the staining characteristics of the major glycoproteins with periodic acid/Schiff's reagent and concanavalin A/peroxidase detection and a combined procedure revealed significant differences in associated carbohydrate structures, and the major concanavalin A-binding component was shown to be GPIIIa. These observations are discussed in the context of functional activities of both membrane systems in the physiological behaviour of the platelet.

scholarly journals Concanavalin A induces interactions between surface glycoproteins and the platelet cytoskeleton.

1982 ◽  
Vol 92 (2) ◽  
pp. 565-573 ◽  
Author(s):  
R G Painter ◽  
M Ginsberg
Keyword(s):  
Concanavalin A ◽  
Surface Membrane ◽  
Dnase I ◽  
Actin Binding ◽  
Resting Cells ◽  
Dependent Manner ◽  
Con A ◽  
Intact Cells ◽  
Platelet Surface ◽  
Surface Glycoproteins

We have measured the association of platelet surface membrane proteins with Triton X-100 (Triton)-insoluble residues in platelets surface labeled with 125I. In both concanavalin A (Con A)-stimulated and resting platelets, this fraction is composed largely of polypeptides with apparent molecular weights of 45,000, 200,000, and 250,000 which comigrate with authentic actin, myosin heavy chain, and actin binding protein, respectively, as judged by PAGE in SDS. Less than 10% of the two major 125I-labeled surface glycoproteins, GPiib and GPIII, were associated with the Triton residue in resting platelets. Within 45 s after Con A addition, 80-95% of these two glycoproteins became associated with the Triton residue and the amount of sedimentable actin doubled. No cosedimentation of GPIIb and III with the cytoskeletal protein-containing Triton residue was seen when Con A was added to a Triton extract of resting cells, indicating that the sedimentation of GPIIb and III seen in Con A-stimulated platelets was not due to precipitation of the glycoproteins by Con A after detergent lysis. Treatment of Triton extracts of Con A-stimulated platelets with DNase I (deoxyribonucleate 5'-oligonucleotidido-hydrolase [EC 3.1.4.5]) inhibited the sedimentation of actin and the two surface glycoproteins in a dose-dependent manner. This inhibition of cosedimentation was not due to an effect of DNase I on Con A-glycoprotein interactions since these two glycoproteins could be quantitatively recovered by Con A-Sepharose affinity absorption in the presence of DNase I. When the Con A bound to the Triton residue was localized ultrastructurally, it was associated with cell-sized structures containing filamentous material. In intact cells, there was simultaneous immunofluorescent coredistribution of surface-bound Con A and myosin under conditions which induced a redistribution of platelet myosin. These data suggest that Con A can, in the intact platelet, induce physical interactions between certain surface glycoproteins and the internal cytoskeleton.

scholarly journals The Characterization of Pig Platelet Membrane Proteins: Studies on Membrane-Associated Actin and the Surface-Associated Phosphodiesterase Activity

1977 ◽  
Author(s):  
D. G. Taylor
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Surface Membrane ◽  
Polyacrylamide Gel ◽  
Molecular Weight Range ◽  
Platelet Surface ◽  
Prominent Component ◽  
Triton X ◽  
Sepharose 4B

Analysis of the polypeptides of a pig platelet surface membrane fraction by SDS-polyacryl-amide gel electrophoresis revealed the presence of approx. 12 components (molecular weight range 12000–200000).A component of 43–45,000 daltons was particularly prominent and this has been identified as actin, and shown to be similar in many respects to the cytoplasmic protein. The membrane-associated actin has been isolated as a pure band by preparative SDS-polyacrylamide gel electrophoresis and analysis of its amino acid composition, although very similar to that of actin purified from whole platelets, showed a much lower content of 3-methylhistidine (approx. 0.05 residues/mole compared with 0.62 residues/mole). The surface membrane-associated phosphodiesterase (towards bis-(p-nitropheny1) phosphate) has also been partially characterised. This enzyme activity can be completely solubilised from the membrane using 0.5% Triton X-100, and can then be isolated as a single peak on Sepharose 4B. The separated enzyme fraction showed considerable purification over the original membrane as illustrated by SDS-polyacrylamide gel electrophoresis with the prominent component being the 95,000 dalton polypeptide. Our studies have also shown that this phosphodiesterase is inhibited competitively by ADP and ATP.

Glanzmann’s Thrombasthenia : Surface Labelling by Various Techniques and Analysis by Gel Electrophoresis.

1979 ◽  
Author(s):  
J.L. McGregor ◽  
K.J. Clemetson ◽  
E. James ◽  
M. Dechavanne
Keyword(s):  
Sialic Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Surface Membrane ◽  
Polyacrylamide Gel ◽  
Galactose Oxidase ◽  
Membrane Glycoproteins ◽  
Platelet Surface ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia

The surface membrane glycoproteins of glanzmann’s thrombasthenia and normal whole platelets were labelled by techniques specific for either sugar or protein moieties. The labelled platelets were solubilized and electrophoresed in reduced or unreduced state by discontinuous SDS-polyacrylamide gel electrophoresis. Galactose oxidase + NaB3H4, labelling, showed with reduced samples 4 glycoprotein bands : a high M.W. glycoprotein and GP Ia, GP Ib, GP IIIb, more intensely labelled than with control platelets but with similar M.W. After treatment with neuraminidase + galactose oxidase + NaB3H4 to remove terminal sialic acid and label penultimate galactose residues the gels showed on both unreduced and reduced samples the absence of PG IIb and IIIa and a relatively broad and intensely labelled GPIb band compared with control platelets. The use of sodium periodate + NaB3H4, to label predominantly sialic acid moieties gave essentially the same number of GP bands in both reduced and unreduced samples as in normal platelets. Lactoperoxidase iodination showed in thrombasthenic platelets both in the reduced and unreduced states the absence of GPIIb, GPIIIa and more intensely labelled GPIb and CPIIIb than with control platelets. The combination of multilabelling and discontinuous Polyacrylamide gel system provides a reliable method for investigating the platelet surface.

scholarly journals Studies on the bivalent-cation-activated ATPase activities of highly purified human platelet surface and intracellular membranes

1986 ◽  
Vol 233 (3) ◽  
pp. 661-668 ◽  
Author(s):  
N Hack ◽  
M Croset ◽  
N Crawford
Keyword(s):  
Atpase Activity ◽  
Blood Platelets ◽  
Present Report ◽  
Surface Membrane ◽  
Storage Site ◽  
Intracellular Membrane ◽  
Bivalent Cation ◽  
Platelet Surface ◽  
Intracellular Membranes ◽  
Dependent Transport

Membrane-bound Ca2+-ATPases are responsible for the energy-dependent transport of Ca2+ across membrane barriers against concentration gradients. Such enzymes have been identified in sarcoplasmic reticulum of muscle tissues and in non-muscle cells in both surface membranes and endoplasmic-reticulum-like intracellular membrane complexes. In a previous study using membrane fractionation by density-gradient and free-flow electrophoresis, we reported that the intracellular membranes of human blood platelets were a major storage site for Ca2+ and involved in maintaining low cytosol [Ca2+] in the unactivated cell. In the present report we demonstrated that the intracellular membranes also exhibit a high-affinity Ca2+-ATPase which appears to be kinetically associated with the Ca2+-sequestering process. We found that both the surface membrane and the intracellular membrane exhibited a basal Mg2+-ATPase activity, but Ca2+ activation of this enzyme was confined only to the intracellular membrane. Use of Ca2+-EGTA buffers to control the extravesicle [Ca2+] allowed a direct comparison of the Ca2+-ATPase and the Ca2+-uptake process over a Ca2+ range of 0.01 microM to 1.0 mM, and it was found that both properties were maximally expressed in the range of external [Ca2+] 1-50 microM, with concentrations greater than 100 microM showing substantial inhibition. Double-reciprocal plots for the Ca2+-ATPase activity and Ca2+ uptake gave apparent Km values for Ca2+ of 0.15 and 0.13 microM respectively. However, similar plots for ATP with the enzyme revealed a discontinuity (two affinity sites, with Km 20 and 145 microM), whereas plots for the Ca2+ uptake gave a single Km value for Ca2+, 1.1 microM. Phosphorylation studies during Ca2+ uptake using [gamma-32P]ATP revealed two components of 90 and 95 kDa phosphorylated at extravesicle [Ca2+] of 3 microM. The Ca2+-ATPase activity, Ca2+ uptake and phosphorylation were all almost completely inhibited in the presence of 500 microM-Ca2+. Similar studies using mixed membranes revealed four other phosphoproteins (50, 40, 20 and 18 kDa) formed in addition to the 90 and 95 kDa components. The findings are discussed in the context of platelet Ca2+ mobilization for function and the mechanisms whereby Ca2+ homoeostasis is controlled in the unactivated cell.

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.

Additional Glycoprotein Defects In Glanzmann’s Thrombasthenia Platelets

1981 ◽  
Author(s):  
J L McGregor ◽  
K J Clemetson ◽  
E James ◽  
A Capitanio ◽  
M Dechavanne ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Gel Electrophoresis ◽  
Lens Culinaris ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Platelet Surface ◽  
Glanzmann’S Thrombasthenia ◽  
Healthy Donors ◽  
Glanzmann's Thrombasthenia ◽  
Dimensional Electrophoresis

Glanzmann’s thrombasthenia (G.T.) platelets are deficient in 2 major membrane GP (IIb and IIIa). In order to investigate if these are the only defects in this disorder, platelets from G.T. patients and from healthy donors were isolated, washed and surface-labelled by techniques specific for protein or for sugars (sialic acid or penultimate galactose/N-acetylgalactosamine residues). Labelled or unlabelled platelets were solubilized in sodium dodecyl sulphate (SDS) and separated by 2-dimensional polyacrylamide gel electrophoresis, first according to isoelectric point and then according to molecular weight. Glycoproteins from unlabelled platelets separated by 2-dimensional electrophoresis were identified by binding of 125I-labelled Lens culinaris lectin (mannose, glucose specific) GPIIbA1 and IIIaA1 were absent in one G.T. patient while in others lower amounts of 2 GP were found in positions similar to these GP. Major membrane GP (IbA1, IbA2, IbB1 and IIIbA1) had more intensely labelled terminal sialic acid moieties in G.T. platelets than in normals. A major membrane GP designated Ic had an altered pi and its penultimate galactose/N-acetyl galactosamine residues labelled more intensely in G.T. platelets than in controls. One high M.Wt. GP and a number of lower M.Wt. GP (IVa, IVb and VII) normally found in platelets of healthy donors were absent in G.T. platelets. These results indicate strongly that there is a major perturbation of the platelet surface in G.T.

scholarly journals Studies on Platelet Glycoproteins by Surface Labeling and SDS Polyacrylamide Gel Electrophoresis

1977 ◽  
Author(s):  
I. Hagen ◽  
N.O. Solum ◽  
M. Peterka
Keyword(s):  
Electrophoretic Mobility ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Conformational State ◽  
Polyacrylamide Gel ◽  
The Other ◽  
Metabolic Inhibitors ◽  
Conventional System ◽  
Platelet Surface ◽  
Release Reaction

Platelet surface (glyco)proteins, and alterations in these in connection with the thrombin-induced release reaction has been studied. Platelets were labeled by lactoperoxidase-catalyzed iodination, and examined by SDS gel electrophoresis in two different gel systems, one conventional(J. Biol. Chem.1969 244 4406), and the other containing urea and EDTA in the gels. In the conventional system the bulk of radioactivity coincided with a PAS band (GP III) of MW about 100, 000. In the other system, the main radioactive peak appeared in the GP II area (MW 120,000), and a shift in the PAS stain intensity from GP III to GP II was seen. Thrombasthenic platelets showed only traces of the GP II band in both systems. The bulk of radioactivity was associated with the surface glycopolypeptide GPS, which is present, but not labeled in normal platelets. In thrombin-released platelets, GPS in its unreduced state has an altered electrophoretic mobility compared to control platelets and platelets which have been incubated with metabolic inhibitors to prevent secretion. The findings indicate that the GP III band consists of two different polypeptides, one of which appears in the GP II area in gels containing urea and EDTA. Further, that thrombasthenic platelet membrane exists in a conformational state different from that of normal platelets. And finally, GPS is in some way involved in, or influenced by, the thrombin-induced release reaction.

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 Two-step affinity-chromatographic purification of cathepsin D from pig myometrium with high yield

1981 ◽  
Vol 197 (2) ◽  
pp. 519-522 ◽  
Author(s):  
E G Afting ◽  
M L Recker
Keyword(s):  
Molecular Weight ◽  
Concanavalin A ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Cathepsin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Yield ◽  
Acid Proteinase ◽  
Chromatographic Purification

Cathepsin D was purified by two-step affinity chromatography on concanavalin A- and pepstatin-Sepharose. The main purification was achieved by washing the enzyme bound to the pepstatin-Sepharose column with buffered 6 M-urea. This step separated cathepsin D from all low- and high-molecular-weight impurities. Although the 1700-fold purified acid proteinase was homogeneous on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, it still showed microheterogeneity.

Studies of the human testis. XIX

1985 ◽  
Vol 108 (2) ◽  
pp. 284-288
Author(s):  
Abdul Waheed ◽  
Stephen J. Winters ◽  
George M. Farrow ◽  
Hiroyuki Oshima ◽  
Philip Troen
Keyword(s):  
Concanavalin A ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Gel Filtration ◽  
Human Testis ◽  
Ammonium Sulphate Precipitation ◽  
Titration Curves ◽  
Androgen Binding Protein ◽  
Androgen Binding

Abstract. Human testosterone-oestradiol-binding globulin (hTeBG) was purified from pregnancy serum by ammonium sulphate precipitation, preparative flatbed electrofocussing, Concanavalin A-Sepharose affinity chromatography, Sephadex G-150 gel filtration, DEAE-Sephadex chromatography and preparative polyacrylamide gel electrophoresis. The yield was 0.3 mg of hTeBG with a specific acitivity of 1.1 nmoles DHT bound per mg. An antiserum to TeBG was raised in rabbits. Anti-hTeBG IgG was separated from rabbit TeBG by DEAE-Affi-Gel-Blue chromatography. Anti-hTeBG was titrated using protein A-Sepharose which quantitatively binds IgG and therefore bound [3H]DHT-hTeBG-anti-TeBG complexes. The androgen binding components from human testis were separated on Concanavalin A-Sepharose columns into excluded and retained fractions. The antibody bound both testis fractions with titration curves which paralleled that of TeBG, indicating that these androphilic proteins share common immunodeterminants with hTe-BG. The possibility that these testicular proteins are identical in amino acid sequence to TeBG and differ only in carbohydrate content will require further verification. Finally, these results indicate that antibodies to TeBG can be used to study human testicular androgen-binding protein.

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