scholarly journals Identification of a specific glycoprotein ligand for P-selectin (CD62) on myeloid cells.

1992 ◽  
Vol 118 (2) ◽  
pp. 445-456 ◽  
Author(s):  
K L Moore ◽  
N L Stults ◽  
S Diaz ◽  
D F Smith ◽  
R D Cummings ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Myeloid Cells ◽  
Apparent Molecular Weight ◽  
Protein S ◽  
Similar Molecular Weight ◽  
Cell Extracts ◽  
Activated Platelets ◽  
Selectin Ligands ◽  
Reducing Conditions ◽  
Selectin Binding

P-selectin (CD62, GMP-140, PADGEM), a Ca(2+)-dependent lectin on activated platelets and endothelium, functions as a receptor for myeloid cells by interacting with sialylated, fucosylated lactosaminoglycans. P-selectin binds to a limited number of protease-sensitive sites on myeloid cells, but the protein(s) that carry the glycans recognized by P-selectin are unknown. Blotting of neutrophil or HL-60 cell membrane extracts with [125I]P-selectin and affinity chromatography of [3H]glucosamine-labeled HL-60 cell extracts were used to identify P-selectin ligands. A major ligand was identified with an approximately 250,000 M(r) under nonreducing conditions and approximately 120,000 under reducing conditions. Binding of P-selectin to the ligand was Ca2+ dependent and was blocked by mAbs to P-selectin. Brief sialidase digestion of the ligand increased its apparent molecular weight; however, prolonged digestion abolished binding of P-selectin. Peptide:N-glycosidase F treatment reduced the apparent molecular weight of the ligand by approximately 3,000 but did not affect P-selectin binding. Western blot and immunodepletion experiments indicated that the ligand was not lamp-1, lamp-2, or L-selectin, which carry sialyl Le(x), nor was it leukosialin, a heavily sialylated glycoprotein of similar molecular weight. The preferential interaction of the ligand with P-selectin suggests that it may play a role in adhesion of myeloid cells to activated platelets and endothelial cells.

scholarly journals Targeting Selectins Mediated Biological Activities With Multivalent Probes

2021 ◽  
Vol 9 ◽  
Author(s):  
Deepak Ganesh ◽  
Prashant Jain ◽  
Chethan Devanur Shanthamurthy ◽  
Suraj Toraskar ◽  
Raghavendra Kikkeri
Keyword(s):  
Tumor Metastasis ◽  
Inflammatory Diseases ◽  
Biological Activities ◽  
Type I ◽  
Blood Capillaries ◽  
Activated Platelets ◽  
Selectin Ligands ◽  
Different Types ◽  
Selectin Binding ◽  
Sialyl Lewisa

Selectins are type-I transmembrane glycoproteins that are ubiquitously expressed on activated platelets, endothelial cells, and leukocytes. They bind to cell surface glycoproteins and extracellular matrix ligands, regulate the rolling of leukocytes in the blood capillaries, and recruit them to inflammatory sites. Hence, they are potential markers for the early detection and inhibition of inflammatory diseases, thrombosis, cardiovascular disorders, and tumor metastasis. Fucosylated and sialylated glycans, such as sialyl Lewisx, its isoform sialyl Lewisa, and heparan sulfate, are primary selectin ligands. Functionalization of these selectin-binding ligands on multivalent probes, such as nanoparticles, liposomes, and polymers, not only inhibits selectin-mediated biological activity but is also involved in direct imaging of the inflammation site. This review briefly summarizes the selectin-mediated various diseases such as thrombosis, cancer and recent progress in the different types of multivalent probes used to target selectins.

scholarly journals Identification of a Reactivating Factor for Adenosylcobalamin-Dependent Ethanolamine Ammonia Lyase

2004 ◽  
Vol 186 (20) ◽  
pp. 6845-6854 ◽  
Author(s):  
Koichi Mori ◽  
Reiko Bando ◽  
Naoki Hieda ◽  
Tetsuo Toraya
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Protein S ◽  
Amino Acid Residues ◽  
Permeabilized Cells ◽  
E Coli ◽  
Cell Extracts ◽  
Auxiliary Protein

ABSTRACT The holoenzyme of adenosylcobalamin-dependent ethanolamine ammonia lyase undergoes suicidal inactivation during catalysis as well as inactivation in the absence of substrate. The inactivation involves the irreversible cleavage of the Co-C bond of the coenzyme. We found that the inactivated holoenzyme undergoes rapid and continuous reactivation in the presence of ATP, Mg2+, and free adenosylcobalamin in permeabilized cells (in situ), homogenate, and cell extracts of Escherichia coli. The reactivation was observed in the permeabilized E. coli cells carrying a plasmid containing the E. coli eut operon as well. From coexpression experiments, it was demonstrated that the eutA gene, adjacent to the 5′ end of ethanolamine ammonia lyase genes (eutBC), is essential for reactivation. It encodes a polypeptide consisting of 467 amino acid residues with predicted molecular weight of 49,599. No evidence was obtained that shows the presence of the auxiliary protein(s) potentiating the reactivation or associating with EutA. It was demonstrated with purified recombinant EutA that both the suicidally inactivated and O2-inactivated holoethanolamine ammonia lyase underwent rapid reactivation in vitro by EutA in the presence of adenosylcobalamin, ATP, and Mg2+. The inactive enzyme-cyanocobalamin complex was also activated in situ and in vitro by EutA under the same conditions. Thus, it was concluded that EutA is the only component of the reactivating factor for ethanolamine ammonia lyase and that reactivation and activation occur through the exchange of modified coenzyme for free intact adenosylcobalamin.

scholarly journals Studies on the endogenous galactose-binding lectin during early development of the embryo of Xenopus laevis

1985 ◽  
Vol 79 (1) ◽  
pp. 105-117
Author(s):  
H. Harris ◽  
S.E. Zalik
Keyword(s):  
Molecular Weight ◽  
Xenopus Laevis ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Aqueous Suspensions ◽  
Reducing Conditions ◽  
Sds Page ◽  
Haemagglutinating Activity ◽  
Chloroform Methanol

Embryos of the frog Xenopus laevis at cleavage, blastula, gastrula and neurula stages contain a galactose-specific lectin. Extracts of gastrula embryos show the highest specific activity for this lectin compared to the other stages. Haemagglutinating activity of crude extracts is inhibited by lactose, alpha-galactose, beta-galactose, alpha Gal(1----4) beta Gal, beta Gal(1----3) alpha GalNAc, beta Gal(1----3) beta GlcNAc, beta Gal (1----4) beta GlcNAc, and most effectively by the disaccharide alpha Gal(1----3) beta Gal. Lectin from all stages was purified by absorption to galactose-linked immunoadsorbent or by affinity chromatography on a column of p-aminophenyl-beta-D-lactoside coupled to Sepharose 4B. In order to identify a single lectin band under reducing conditions in sodium dodecyl sulphate/polyacrylamide electrophoresis SDS/PAGE, it was necessary to treat aqueous suspensions of the purified lectin with chloroform/methanol (2:1, v/v). The lectin remained in the aqueous layer and gave rise on SDS/PAGE to a distinct band of 65 500 +/− 2780 molecular weight. Aqueous suspensions of the purified lectin that were not subjected to extraction with chloroform/methanol gave rise to several bands. Isoelectric focussing of the purified lectin resulted in two bands that separated at pI 4.3 and 4.5. In aqueous solution in the presence of lactose the chloroform/methanol-treated lectin appears to be an aggregate of apparent molecular weight of 375 000; the non-treated lectin under the same conditions has an apparent molecular weight of 490 000.

Comparison of the binding of 59Fe- and 239Pu-transferrin to rat liver cell membranes

1990 ◽  
Vol 9 (1) ◽  
pp. 17-24 ◽  
Author(s):  
F. Planas-Bohne ◽  
W. Rau
Keyword(s):  
Molecular Weight ◽  
Cell Membrane ◽  
Membrane Protein ◽  
Rat Liver ◽  
Liver Cell ◽  
Cell Membranes ◽  
Apparent Molecular Weight ◽  
Protein S ◽  
Membrane Protein Complex ◽  
Isolated Cell

The binding of the 59Fe and 239Pu complexes of transferrin and 125I labelled transferrin [Tf (125I) ] to isolated cell membranes of rat liver has been studied. Transferrin forms a complex with an integral protein of the membrane which has an apparent molecular weight of about 180 kDa and is stable only at pH 7.4. Iron-59 is eluted from Sephacryl S 300 columns together with Tf (125I) or the Tf-membrane protein complex while 239Pu seems to be bound to different membrane protein(s). After isolation of the Tf-binding protein from 35S-labelled membranes and incubation with one of the metal-Tf complexes 59Fe elutes from a Sephacryl S 300 column together with 35S at an apparent molecular weight of ca. 250 kDa while 239Pu is found in fractions of lower molecular weight. It is concluded from these results that there are Tf-receptors in the liver cell membrane to which iron transferrin may bind. Plutonium, however, seems to be dissociated from Tf and bound directly to other membrane proteins.

scholarly journals Nonlytic simian virus 40-specific 100K phosphoprotein is associated with anchorage-independent growth in simian virus 40-transformed and revertant mouse cell lines.

1981 ◽  
Vol 1 (11) ◽  
pp. 994-1006 ◽  
Author(s):  
S Chen ◽  
M Verderame ◽  
A Lo ◽  
R Pollack
Keyword(s):  
Molecular Weight ◽  
Cell Lines ◽  
Rank Correlation ◽  
Simian Virus 40 ◽  
Apparent Molecular Weight ◽  
Simian Virus ◽  
T Antigen ◽  
Viral Gene ◽  
Semisolid Medium ◽  
Cell Extracts

Normal fibroblasts display two distinct growth controls which can be assayed as requirements for serum or for anchorage. Interaction of mouse 3T3 fibroblasts with simian virus 40 (SV40) thus generates four classes of transformed cells. We have examined viral gene expression in these four classes of cell lines. Immunoprecipitation of [35S]methionine-labeled cell extracts with an antiserum obtained from tumor-bearing hamsters detected the SV40 large T and small t proteins (94,000 molecular weight [94K], 17K) and the nonviral host 54K protein in all cell lines tested. A tumor antigen with an apparent molecular weight of 100,000 was also found in some, but not all, lines. Similar "super T" molecules have been found by others in many rodent transformed lines. We carried out an analysis of the relation of phenotype to relative amounts of these proteins in cell lines of the four classes, using the Spearman rank correlation test. The amount of the 100K T antigen relative to the 94K T antigen or to total viral protein was well correlated with the ability to form colonies in semisolid medium. No significant correlation was found between quantities of labeled 94K T antigen, 54K host antigen, or 17K t antigen and either serum or anchorage independence. Mouse cells transformed with the small t SV40 deletion mutant 884 synthesized a 100K T antigen, suggesting that small t is not required for the production of this protein. The 100K T antigen migrated more slowly than lytic T. Since mixtures of extracts from cells expressing and lacking the 100K T antigen yielded the expected amount of this protein, it is unlikely that the 100K T derives from the 94K protein by a posttranslational modification.

Megakaryocytic cells synthesize and platelets secrete α5-laminins, and the endothelial laminin isoform laminin 10 (α5β1γ1) strongly promotes adhesion but not activation of platelets

2006 ◽  
Vol 95 (01) ◽  
pp. 85-93 ◽  
Author(s):  
Ayele Nigatu ◽  
Wondossen Sime ◽  
Gezahegn Gorfu ◽  
Tarekegn Geberhiwot ◽  
Ingegerd Andurén ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Blood Cells ◽  
Blood Platelets ◽  
Apparent Molecular Weight ◽  
Cell Aggregation ◽  
Adhesive Properties ◽  
Circulating Cells ◽  
Activated Platelets ◽  
Intracellular Expression

SummaryFollowing vascular injury, basement membrane (BM) components of the blood vessels are exposed to circulating cells and may contribute to hemostasis and/or thrombosis. Laminins 8 (LN-8) (α4β1γ1) and 10 (LN-10) (α5β1γ1) are major laminin isoforms of the endothelial BM, and LN-8 is also secreted by activated platelets. In the present study, we demonstrate synthesis of α5-laminins LN-10 and LN-11 (α5β2γ1) by megakaryocytic cells, and intracellular expression of these laminin isoforms in blood platelets. In contrast to platelet LNα4 chain that had an apparent molecular weight of 180 kDa and associated mostly to LNβ1 chain, platelet LNα5 consisted of 300/350 kDa polypeptides and associated mainly to LNβ2. Both α4– and α5-laminins were secreted by platelets following stimulation. When compared to recombinant human (rh) LN-8, rhLN-10 was much more adhesive to platelets, though adhesion to both proteins was largely mediated via α6β1 integrin. In spite of their adhesive properties, rhLN-8 and rhLN-10 induced neither P-selectin expression nor cell aggregation, two signs of platelet activation. This study demonstrates synthesis/expression of heterotrimeric α5-laminins in hematopoietic/blood cells, and provides evidence for the adhesive, but not activating, role of endothelial laminin isoforms in platelet biology.

Waterlogging and soil reduction affect the amount and apparent molecular weight distribution of dissolved organic matter in wetland soil: a laboratory study

Soil Research ◽  
2018 ◽  
Vol 56 (1) ◽  
pp. 28 ◽  
Author(s):  
Asmaa Rouwane ◽  
Malgorzata Grybos ◽  
Isabelle Bourven ◽  
Marion Rabiet ◽  
Gilles Guibaud
Keyword(s):  
Molecular Weight ◽  
Organic Matter ◽  
Dissolved Organic Matter ◽  
Humic Substances ◽  
Apparent Molecular Weight ◽  
Redox Condition ◽  
Redox Conditions ◽  
Wetland Soil ◽  
Soil Redox ◽  
Reducing Conditions

The release of dissolved organic matter (DOM) from wetland soils is an important pathway for the input of organic compounds into adjacent aquatic environments. In the present study we investigated, under controlled laboratory conditions, the quantity and quality of DOM released from a wetland soil subject to waterlogging and reducing conditions. Three soil redox conditions (oxic, moderately reducing and advanced reducing) were distinguished based on nitrate, ferrous ions and sulfate concentrations in soil solution. Under each redox condition, the quantity (dissolved organic carbon (DOC), humic substances and peptides plus proteins (P-PN) and quality (aromaticity; specific ultraviolet absorbance at 254 nm (SUVA254nm)) and apparent molecular weight (aMW) distribution) of DOM were investigated. The results showed that soil redox condition affects the amount and properties of mobilised DOM. The rate of DOM release and SUVA254 values were highest during the transition from oxic to moderately reducing conditions, whereas both stabilised during progression to advanced reducing conditions. In addition, the mobilised DOM is expected to be more reactive because of an increase in polar substituents in aromatic structures between oxic and moderately reducing conditions. During the development of moderately reducing conditions, dissolved humic substances increased significantly, whereas their aMW distribution (between 500 and 6000 ) remained constant for each of the three different redox conditions. In contrast, the quantity of dissolved P-PN remained low and steady under the three redox conditions, whereas the aMW distribution of protein-like and microbial by-product-like compounds decreased during the development of reducing conditions (aMW of compounds between 100 and >100 000).

Nonlytic simian virus 40-specific 100K phosphoprotein is associated with anchorage-independent growth in simian virus 40-transformed and revertant mouse cell lines

1981 ◽  
Vol 1 (11) ◽  
pp. 994-1006
Author(s):  
S Chen ◽  
M Verderame ◽  
A Lo ◽  
R Pollack
Keyword(s):  
Molecular Weight ◽  
Cell Lines ◽  
Rank Correlation ◽  
Simian Virus 40 ◽  
Apparent Molecular Weight ◽  
Simian Virus ◽  
T Antigen ◽  
Viral Gene ◽  
Semisolid Medium ◽  
Cell Extracts

Normal fibroblasts display two distinct growth controls which can be assayed as requirements for serum or for anchorage. Interaction of mouse 3T3 fibroblasts with simian virus 40 (SV40) thus generates four classes of transformed cells. We have examined viral gene expression in these four classes of cell lines. Immunoprecipitation of [35S]methionine-labeled cell extracts with an antiserum obtained from tumor-bearing hamsters detected the SV40 large T and small t proteins (94,000 molecular weight [94K], 17K) and the nonviral host 54K protein in all cell lines tested. A tumor antigen with an apparent molecular weight of 100,000 was also found in some, but not all, lines. Similar "super T" molecules have been found by others in many rodent transformed lines. We carried out an analysis of the relation of phenotype to relative amounts of these proteins in cell lines of the four classes, using the Spearman rank correlation test. The amount of the 100K T antigen relative to the 94K T antigen or to total viral protein was well correlated with the ability to form colonies in semisolid medium. No significant correlation was found between quantities of labeled 94K T antigen, 54K host antigen, or 17K t antigen and either serum or anchorage independence. Mouse cells transformed with the small t SV40 deletion mutant 884 synthesized a 100K T antigen, suggesting that small t is not required for the production of this protein. The 100K T antigen migrated more slowly than lytic T. Since mixtures of extracts from cells expressing and lacking the 100K T antigen yielded the expected amount of this protein, it is unlikely that the 100K T derives from the 94K protein by a posttranslational modification.

scholarly journals Size characteristics of human leucocyte interferon under reducing and non-reducing conditions

1981 ◽  
Vol 193 (3) ◽  
pp. 947-951 ◽  
Author(s):  
I A Braude ◽  
L S Lin ◽  
W E Stewart
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Polyacrylamide Gel ◽  
Lower Molecular Weight ◽  
Reducing Conditions ◽  
Size Heterogeneity

Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was used to characterize human leucocyte interferon (HuIFN-alpha) under reducing and non-reducing conditions. Under non-reducing conditions HuIFN-alpha possesses two size forms, but under reducing conditions (r-HuIFN-alpha) only one is observed. The apparent molecular weight of this one form varies with the concentration of 2-mercaptoethanol used. When r-HuIFN-alpha is permitted to reoxidize the bimodal configuration of HuIFN-alpha is restored. The size heterogeneity of native HuIFN-alpha can be eliminated by mild treatment with NaIO4 [HuIFN-alpha/IO4; Stewart II, Lin, Wiranowska-Stewart & Cantell (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 4200-4204]. The size of the HuIFN-alpha/IO4 increases after treatment with 2-mercaptoethanol (r-HuIFN-alpha/IO4) and the apparent molecular weight of this component also varies with the concentration of 2-mercaptoethanol used. In the case of r-HuIFN-alpha the single peak observed apparently originates from both the higher- and lower-molecular-weight components.

scholarly journals Monospecific and common glycoprotein ligands for E- and P-selectin on myeloid cells.

1994 ◽  
Vol 125 (2) ◽  
pp. 471-481 ◽  
Author(s):  
M Lenter ◽  
A Levinovitz ◽  
S Isenmann ◽  
D Vestweber
Keyword(s):  
Sialic Acid ◽  
Cell Adhesion Molecules ◽  
Recombinant Antibody ◽  
Myeloid Cells ◽  
Cell Binding ◽  
Hl60 Cells ◽  
Selectin Ligands ◽  
Mature Mouse ◽  
Selectin Ligand ◽  
Selectin Binding

E- and P-selectin are inducible cell adhesion molecules on endothelial cells, which function as Ca(2+)-dependent lectins and mediate the binding of neutrophils and monocytes. We have recently identified a 150-kD glycoprotein ligand for E-selectin on mouse myeloid cells, using a recombinant antibody-like form of mouse E-selectin. Here, we report that this ligand does not bind to an analogous P-selectin fusion protein. Instead, the chimeric P-selectin-IgG protein recognizes a 160-kD glycoprotein on the mouse neutrophil progenitor 32D cl 3, on mature mouse neutrophils and on human HL60 cells. The binding is Ca(2+)-dependent and requires the presence of sialic acid on the ligand. This P-selectin-ligand is not recognized by E-selectin. Removal of N-linked carbohydrate side chains from the 150-kD and the 160-kD monospecific selectin ligands abolishes the binding of both ligands to the respective selectin. Treatment of HL60 cells with Peptide: N-glycosidase F inhibited cell binding to P- and E-selectin. In addition, glycoproteins of 230 and 130 kD were found on mature mouse neutrophils, which bound both to E- and P-selectin in a Ca(2+)-dependent fashion. The signals detected for these ligands were 15-20-fold weaker than those for the monospecific ligands. Both proteins were heavily sialylated and selectin-binding was blocked by removal of sialic acid, but not by removal of N-linked carbohydrates. Our data reveal that E- and P-selectin recognize two categories of glycoprotein ligands: one type requires N-linked carbohydrates for binding and is monospecific for each of the two selectins and the other type binds independent of N-linked carbohydrates and is common for both endothelial selectins.

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