scholarly journals Purification and biochemical properties of a high-molecular-mass inositol 1,4,5-trisphosphate 3-kinase isoenzyme in human platelets

1994 ◽  
Vol 298 (3) ◽  
pp. 669-673 ◽  
Author(s):  
D Communi ◽  
V Vanweyenberg ◽  
C Erneux
Keyword(s):  
Molecular Mass ◽  
Specific Activity ◽  
Human Platelet ◽  
Biochemical Properties ◽  
Human Platelets ◽  
High Molecular Mass ◽  
Sds Page ◽  
Inositol 1,4,5 Trisphosphate ◽  
Rapid Purification ◽  
Kinase A

The phosphorylation of inositol 1,4,5-trisphosphate (InsP3) to inositol 1,3,4,5-tetrakisphosphate (InsP4) is catalysed by InsP3 3-kinase. A method is presented for a rapid purification of the enzyme from human platelets. The purified enzyme was identified as a polypeptide of M(r) 69,000-70,000 after SDS/PAGE. It had a specific activity of 1.45 +/- 0.1 mumol/min per mg, and the degree of stimulation by Ca2+/calmodulin was 17-fold at saturating calmodulin and 10 microM free Ca2+. The Km for InsP3 and for ATP was 2.0 microM and 2.5 mM respectively. Human platelet InsP3 3-kinase was not recognized by immunodetection with anti-(InsP3 3-kinase A) or anti-(InsP3 3-kinase B) antibodies. These data provide the first biochemical evidence for the existence of a novel InsP3 3-kinase isoenzyme in human platelets, which is distinct from previously reported InsP3 3-kinase A and InsP3 3-kinase B.

Studies on the Organisation of Glycoproteins and Proteins in Human Platelet Membranes

1979 ◽  
Author(s):  
A.T. Nurden ◽  
D. Dupuis ◽  
H. de la Baume ◽  
J.P. Caen
Keyword(s):  
Human Platelet ◽  
Membrane Vesicles ◽  
Human Platelets ◽  
Cross Linking ◽  
Platelet Response ◽  
Membrane Glycoproteins ◽  
Sh Groups ◽  
Sds Page ◽  
The Cross ◽  
Neighbour Analysis

Addition of wheat germ agglutinin (WGA) (50 ug/ml) to washed human platelets (3 x 108/ml) resulted in platelet activation and the release of l4C-5HT within the same time scale as 0.05 units/ml thrombin. In contrast, succinyl-WGA (100 ug/ml) induced no platelet response. The increased valency of WGA (4) compared with succinyl-WGA (2) suggests that the activation is induced through the cross-linking (immobilisation ?) of closely associated receptors in the membrane. This finding induced us to attempt to cross-link and thereby identify adjacent molecules in the membrane by “near-neighbour” analysis. Constituent -SH groups were oxidised employing Cu2+/phenanthroline or diamide as catalysts, and polymers formed as a result of intermolecular -S-S- formation between adjacent molecules were identified by SDS-PAGE. Although previous reports have shown that the major human platelet membrane glycoproteins contain -SH groups, no apparent cross-linking of the glycoproteins was located following the incubation of either washed platelets or isolated membranes with Cu2+/phenanthroline or diamide. However bidimensional SDS-PAGE (1st dimension non-reduced, 2nd dimension reduced) showed the presence of several protein polymers including complexes formed by the cross-linking of 3 large polypeptides of M. Wt. 250 000, 220 000 and 200 000. These components were easily eluted from membrane vesicles at pH 10 and may represent closely associated constituents at the cytoplasmic surface of the plasma membrane.

scholarly journals Purification and characterization of a thermostable ADP-glucose pyrophosphorylase from Thermus caldophilus GK-24

1996 ◽  
Vol 319 (3) ◽  
pp. 977-983 ◽  
Author(s):  
Jeong Heon KO ◽  
Cheorl Ho KIM ◽  
Dae-Sil LEE ◽  
Yu Sam KIM
Keyword(s):  
Molecular Mass ◽  
Specific Activity ◽  
Gel Filtration ◽  
Higher Plant ◽  
Sds Page ◽  
Thermus Caldophilus ◽  
Internal Peptide ◽  
Adp Glucose Pyrophosphorylase ◽  
Fructose 1,6 Bisphosphate

An extremely thermostable ADP-glucose pyrophosphorylase (AGPase) has been purified from Thermus caldophilus GK-24 to homogeneity by chromatographic methods, including gel filtration and ion-exchange and affinity chromatography. The specific activity of the enzyme was enriched 134.8-fold with a recovery of 10.5%. The purified enzyme was a single band by SDS/PAGE with a molecular mass of 52 kDa. The homotetrameric structure of the native enzyme was determined by gel filtration analysis, which showed a molecular mass of 230 kDa on a Superose-12 column, indicating that the structure of the enzyme is different from the heterotetrameric structures of higher-plant AGPases. The enzyme was most active at pH 6.0. The activity was maximal at 73–78 °C and its half-life was 30 min at 95 °C. Kinetic and regulatory properties were characterized. It was found that AGPase activity could be stimulated by a number of glycolytic intermediates. Fructose 6-phosphate, fructose 1,6-bisphosphate, phenylglyoxal and glucose 6-phosphate were effective activators, of which fructose 1,6-bisphosphate was the most effective. The enzyme was inhibited by phosphate, AMP or ADP. ATP and glucose 1-phosphate gave hyperbolic-shaped rate-concentration curves in the presence or absence of activator. A remarkable aspect of the amino acid composition was the existence of the hydrophobic and Ala+Gly residues. The N-terminal and internal peptide sequences were determined and compared with known sequences of various sources. It was apparently similar to those of AGPases from other bacterial and plant sources, suggesting that the enzymes are structurally related.

scholarly journals Molecular interaction of the proteasome (multicatalytic proteinase). Evidence that the proteasome is not a constituent of the ‘26 S’ multienzyme complex

1991 ◽  
Vol 280 (1) ◽  
pp. 225-232 ◽  
Author(s):  
A Seelig ◽  
P M Kloetzel ◽  
L Kuehn ◽  
B Dahlmann
Keyword(s):  
Molecular Mass ◽  
Molecular Interaction ◽  
Gradient Centrifugation ◽  
Mass Range ◽  
High Molecular Mass ◽  
Multienzyme Complex ◽  
Multicatalytic Proteinase ◽  
Sds Page ◽  
Specific Antisera ◽  
Rabbit Reticulocytes

On the basis of recent reports that suggested that proteasomes, via an ATP-dependent process, become integral components of a ‘26 S’ complex possessing 3-carboxypropionyl-Leu-Leu-Val-Tyr 4-methylcoumarin-7-ylamide-hydrolysing activity, we have investigated the molecular interaction of proteasomes in ATP-stabilized fraction II (proteins absorbed on DEAE-matrix and eluted with 0.5 M-KCl) of rabbit reticulocytes and mouse liver. Analysis of the various extracts by (NH4)2SO4 fractionation, velocity-gradient centrifugation, non-denaturing PAGE and SDS/PAGE and immunoblotting with proteasome-specific antisera failed to identify the proteasome as part of a higher-molecular-mass ‘26 S’ multienzyme complex. In all instances proteasomes are identified in their ‘free’ 650 kDa ‘20 S’ form. In addition to the proteasome and independent of the presence of MgATP, we isolated a high-molecular-mass proteinase whose electrophoretic migration behaviour and sedimentation rate correspond to that of the previously described ‘26 S’ proteinase. This ‘26 S’ proteinase possesses a strong 3-carboxypropionyl-Leu-Leu-Val-Tyr 4-methylcoumarin-7-ylamide-hydrolysing activity and is composed of several non-identical polypeptides in the molecular-mass range 20-150 kDa. Despite its similarity to proteasomal enzyme activity, protein analysis and immunoblotting experiments demonstrate that neither the intact proteasome nor subunits thereof are components of the ‘26 S’ proteinase complex.

Synthesis and Ultrastructural Localization of Protein C Inhibitor in Human Platelets and Megakaryocytes

Blood ◽  
1999 ◽  
Vol 94 (4) ◽  
pp. 1300-1312
Author(s):  
Maria J. Prendes ◽  
Edith Bielek ◽  
Margareta Zechmeister-Machhart ◽  
Erika Vanyek-Zavadil ◽  
Veronica A. Carroll ◽  
...  
Keyword(s):  
Protein C ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Plasminogen Activator Inhibitor ◽  
Ultrastructural Localization ◽  
Human Platelets ◽  
Plasminogen Activator Inhibitor 1 ◽  
Sds Page ◽  
Protein C Inhibitor

The occurrence of protein C inhibitor (PCI) in human platelets and megakaryocytes was analyzed. As judged from enzyme-linked immunosorbent assays (ELISAs), PCI was present in platelets at a concentration of 160 ng/2 × 109 cells. Its specific activity was 5 times higher than that of plasma PCI. Consistently, mainly the 57-kD form (active PCI) and some high molecular weight (Mr) forms, but no bands corresponding to cleaved PCI, were detected when platelet lysates were immunoprecipitated with monoclonal anti-PCI-IgG and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The localization of PCI in platelets was studied by immunofluorescence histochemistry and immunotransmission electron microscopy: PCI was detected in  granules, in the open canalicular system, and on the plasma membrane. At these sites, colocalization with plasminogen activator inhibitor-1 was seen. Studies were performed to clarify whether platelet PCI is endogenously synthesized or taken up from plasma. Internalization of biotinylated-PCI was analyzed using platelets in suspension and gold-labeled streptavidin for visualization of incorporated biotin. Dose- and time-dependent uptake of PCI was found. PCI mRNA was detected in platelets by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blotting, as well as in megakaryocytes by in situ hybridization of human bone marrow cryosections. We therefore conclude that platelets contain a functionally active PCI pool that is derived from both endogenous synthesis as well as internalization.

scholarly journals Identification of a human gastric mucin precursor: N-linked glycosylation and oligomerization

1994 ◽  
Vol 304 (3) ◽  
pp. 693-698 ◽  
Author(s):  
L W J Klomp ◽  
L van Rens ◽  
G J Strous
Keyword(s):  
Molecular Mass ◽  
Mechanical Damage ◽  
Polyclonal Antiserum ◽  
High Molecular Mass ◽  
Apparent Molecular Mass ◽  
Gastric Mucin ◽  
Gastric Mucus ◽  
Apical Side ◽  
Sds Page ◽  
Glycosylation Inhibitor

Gastric mucin plays an important role in the protection of the stomach wall from chemical, microbiological and mechanical damage. We have previously isolated human gastric mucus glycoproteins and raised a polyclonal antiserum against these macromolecules. This antiserum specifically reacted with gastric mucins in immunoblotting experiments and stained mucous granules at the apical side of gastric surface epithelial cells. A similar staining pattern was obtained after incubation with an antiserum against rat gastric mucin. Next we used the antiserum in pulse-chase experiments of human stomach tissue explants. After short labelling periods with [35S]methionine and [35S]cysteine, the antiserum reacted with a polypeptide with an apparent molecular mass of approx. 500 kDa as determined by SDS/PAGE, which was converted after 90 min into a heterogeneous high-molecular-mass glycoprotein. This high-molecular-mass form, but not the 500 kDa polypeptide, was detectable in the culture medium after 2 h. This strongly suggests that the 500 kDa polypeptide is the precursor of the purified gastric mucin. Analysis of pulse-chase experiments by non-reducing SDS/PAGE revealed that the precursors form disulphide-linked oligomers early in biosynthesis, before the addition of O-linked sugars. After preincubation with the N-glycosylation inhibitor, tunicamycin, the apparent molecular mass of the precursor decreased marginally but consistently, indicating that N-linked glycan chains are present on the mucin precursor.

scholarly journals Rapid purification and identification of calcyphosine, a Ca2+-binding protein phosphorylated by protein kinase A

1995 ◽  
Vol 306 (1) ◽  
pp. 147-151 ◽  
Author(s):  
R Lecocq ◽  
F Lamy ◽  
C Erneux ◽  
J E Dumont
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Conformational Changes ◽  
Hydrophobic Interaction Chromatography ◽  
Peptide Sequencing ◽  
Native Form ◽  
Sds Page ◽  
Dependent Protein ◽  
Rapid Purification ◽  
Kinase A

A method is presented for the rapid purification of dog thyroid calcyphosine, a protein previously identified as a major substrate for cyclic AMP-dependent protein kinase in dog thyroid slices stimulated by thyrotropin [Lecocq, Lamy and Dumont (1979) Eur. J. Biochem. 102, 147-152]. The protein was previously identified as a spot on two-dimensional gels and is now purified in its native form by a procedure involving three chromatographic steps. Homogeneous calcyphosine identified by SDS/PAGE, immunoblotting and peptide sequencing can be obtained within 7 h. As for calmodulin, Ca(2+)-dependent conformational changes can be shown by Ca(2+)-dependent hydrophobic interaction chromatography using phenyl-Sepharose. Unlike calmodulin, calcyphosine is a substrate for protein kinase A.

scholarly journals Biosynthesis of rat MUC2 in colon and its analogy with human MUC2

1995 ◽  
Vol 309 (1) ◽  
pp. 221-229 ◽  
Author(s):  
K M A J Tytgat ◽  
F J Bovelander ◽  
F J M Opdam ◽  
A W C Einerhand ◽  
H A Büller ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Buoyant Density ◽  
Polyclonal Antiserum ◽  
High Molecular Mass ◽  
Rat Colon ◽  
Metabolic Labelling ◽  
Colonic Mucin ◽  
Sds Page ◽  
High Degree ◽  
Degree Of Similarity

In order to identify the mucins synthesized and secreted in the rat colon, we studied their biochemical characteristics and biosynthesis and evaluated their analogy to human colonic mucins. Purified mucin from both species appeared similar with respect to composition, buoyant density and mobility on SDS/PAGE. Isolated rat colonic mucin (RCM) was used to elicit a polyclonal antiserum, which was used in metabolic labelling studies to identify mucins and mucin precursors. RCM is synthesized as a 600 kDa precursor protein, which oligomerizes before O-glycosylation. The mature, high-molecular mass mucin is secreted and displays an anomalous molecular mass on SDS/PAGE of approximately 650 kDa. Polymorphism in precursor size was found among different rats, suggesting genetic heterogeneity. Molecular mass, biosynthesis and secretion of RCM appeared similar to human MUC2. Moreover, RCM precursor could be immunoprecipitated using specific anti-(human MUC2) antisera, indicating that the RCM can be designated rat MUC2. This study describes the biosynthesis of two homologous mucins in two different species. The high degree of similarity suggests functional analogy.

scholarly journals Purification and characterization of human platelet glutathione-S- transferase

Blood ◽  
1986 ◽  
Vol 67 (6) ◽  
pp. 1595-1599
Author(s):  
J Loscalzo ◽  
J Freedman
Keyword(s):  
Specific Activity ◽  
Human Platelet ◽  
Reduced Glutathione ◽  
Human Platelets ◽  
Glutathione S Transferase ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Complete Purification ◽  
High Concentrations

A glutathione-S-transferase was isolated and purified to homogeneity from human platelets. With a combination of ammonium sulfate fractionation and chromatographic methods, 0.2 mg of pure enzyme was obtained from 9 X 10(11) platelets with a 12% recovery. The purified enzyme had a specific activity of 7.5 U per milligram, representing an approximately 1,100-fold purification. The enzyme was found to be anionic, with an isoelectric point of 4.6. With reduced glutathione as a co-substrate, platelet glutathione-S-transferase was most active with the synthetic substrate, 1-chloro-2,4-dinitrobenzene, less active with 1,2-dichloro-4-nitrobenzene, and essentially inactive with nitroglycerin and 1,2-epoxy-3-(p-nitrophenoxy)-propane. The pH optimum for activity with glutathione and 1-chloro-2,4-dinitrobenzene was 7.0. Indomethacin (1-(p-chlorobenzoyl)-5-methoxy-2-methyindole-3-acetic acid), a chlorobenzene derivative, noncompetitively inhibited human platelet glutathione-S-transferase with an apparent KI of 0.23 mmol/L. This study represents the first complete purification and characterization of a glutathione-S-transferase from platelets. The presence of this enzyme in the platelet, within which high concentrations of reduced glutathione coexist, suggests the potential importance of the platelet in detoxification reactions and in the synthesis of the glutathione adducts of leukotriene metabolism.

Synthesis and Ultrastructural Localization of Protein C Inhibitor in Human Platelets and Megakaryocytes

Blood ◽  
1999 ◽  
Vol 94 (4) ◽  
pp. 1300-1312 ◽  
Author(s):  
Maria J. Prendes ◽  
Edith Bielek ◽  
Margareta Zechmeister-Machhart ◽  
Erika Vanyek-Zavadil ◽  
Veronica A. Carroll ◽  
...  
Keyword(s):  
Protein C ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Plasminogen Activator Inhibitor ◽  
Ultrastructural Localization ◽  
Human Platelets ◽  
Plasminogen Activator Inhibitor 1 ◽  
Sds Page ◽  
Protein C Inhibitor

Abstract The occurrence of protein C inhibitor (PCI) in human platelets and megakaryocytes was analyzed. As judged from enzyme-linked immunosorbent assays (ELISAs), PCI was present in platelets at a concentration of 160 ng/2 × 109 cells. Its specific activity was 5 times higher than that of plasma PCI. Consistently, mainly the 57-kD form (active PCI) and some high molecular weight (Mr) forms, but no bands corresponding to cleaved PCI, were detected when platelet lysates were immunoprecipitated with monoclonal anti-PCI-IgG and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The localization of PCI in platelets was studied by immunofluorescence histochemistry and immunotransmission electron microscopy: PCI was detected in  granules, in the open canalicular system, and on the plasma membrane. At these sites, colocalization with plasminogen activator inhibitor-1 was seen. Studies were performed to clarify whether platelet PCI is endogenously synthesized or taken up from plasma. Internalization of biotinylated-PCI was analyzed using platelets in suspension and gold-labeled streptavidin for visualization of incorporated biotin. Dose- and time-dependent uptake of PCI was found. PCI mRNA was detected in platelets by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blotting, as well as in megakaryocytes by in situ hybridization of human bone marrow cryosections. We therefore conclude that platelets contain a functionally active PCI pool that is derived from both endogenous synthesis as well as internalization.

scholarly journals The interleukin-1 receptor in Raji human B-lymphoma cells. Molecular characterization and evidence for receptor-mediated activation of gene expression

1989 ◽  
Vol 260 (3) ◽  
pp. 657-663 ◽  
Author(s):  
R Horuk ◽  
J A McCubrey
Keyword(s):  
Molecular Mass ◽  
Interleukin 1 ◽  
Polyacrylamide Gel Electrophoresis ◽  
Kinetic Studies ◽  
Cell Types ◽  
Biochemical Properties ◽  
Dissociation Rate ◽  
Raji Cells ◽  
B Lymphoma Cells ◽  
Sds Page

In a previous paper [Horuk, Huang, Covington & Newton (1987) J. Biol. Chem. 262, 16275-16278] we reported that there were fundamental differences in the biochemical properties of the interleukin-1 (IL-1) receptor between Raji and EL4 cell lines. In the present study we have investigated the basis for these differences. Kinetic studies measuring the on and off rates of IL-1 receptor binding revealed that the low-affinity IL-1-binding sites observed in Raji cells, compared with EL4 cells, result from a combination of a lower association rate and a higher dissociation rate in the Raji cells. The turnover of the Raji IL-1 receptor, measured by inhibiting protein synthesis with cycloheximide, was much faster than that of the EL4 IL-1 receptor, with a half-time of 2 h as against 5 h. Treatment of 125I-IL-1-labelled IL-1 receptors in Raji and EL4 cells with neuraminidase decreased their molecular mass by approx. 2-5 kDa as assessed by SDS/polyacrylamide-gel electrophoresis (PAGE). The covalently labelled IL-1 receptors in both cell types were sensitive to treatment with endoglycosidase F, which decreased their molecular mass on SDS/PAGE by 12-13 kDa. Incubation of Raji cells with maximally stimulating doses of IL-1 resulted in an increase in the nascent RNA levels of several genes, including the IL-2 receptor and the proto-oncogenes c-Ha-ras and c-myc.

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