Identification of albumin-binding proteins of thymocyte plasmalemma

1996 ◽  
Vol 16 (5) ◽  
pp. 425-438
Author(s):  
Ludy Dorbrila ◽  
Geo Serban ◽  
Constantina Heltianu
Keyword(s):  
Plasma Membrane ◽  
Affinity Chromatography ◽  
Binding Proteins ◽  
Specific Binding ◽  
Membrane Surface ◽  
Albumin Binding ◽  
Differential Centrifugation ◽  
Ligand Affinity ◽  
Agarose Beads ◽  
Sds Page

In the present work we examined whether the interaction between albumin molecules and thymocytes involves albumin-binding proteins (ABP). Two plasmalemma-rich fractions obtained by differential centrifugation from rat thymus lymphocytes were characterized biochemically and morphologically. These fractions were examined by ligand-blotting and ligand affinity chromatography techniques. Plasmalemma proteins separated by SDS-PAGE were electrotransferred onto nitrocellulose membranes and incubated with125I-albumin, in the presence or absence of excess native albumin. The autoradiogram revealed specific binding to two sets of polypeptides of 16–18 and 29–31 kDa, which could be blocked by native albumin. To elucidate whether albumin-binding proteins are exposed on the cell surface, intact lymphocytes were surface radioiodinated and membrane fractions prepared from them were subjected to affinity chromatography on albumin-agarose beads. The proteins thus purified had, like ABP, Mr of 16 and 31. These data indicate that ABP (i) are components of thymocyte plasma membrane, (ii) have apparent molecular mass of 16–18 and 29–31 kDa, and (iii) are exposed on the outer membrane surface.

scholarly journals Heparin- and Zn2+-binding proteins from boar seminal plasma.

1999 ◽  
Vol 46 (4) ◽  
pp. 935-939 ◽  
Author(s):  
D Hołody ◽  
J Strzezek
Keyword(s):  
Amino Acid ◽  
Affinity Chromatography ◽  
Molecular Mass ◽  
Seminal Plasma ◽  
Binding Proteins ◽  
Reproductive Tract ◽  
Female Reproductive Tract ◽  
Heparin Binding ◽  
Sds Page ◽  
Protein Components

Low molecular mass, heparin-binding proteins from seminal plasma play an important role in gametes interaction whereas plasmatic Zn2+-binding proteins stabilize chromatin and plasmalemma structures and protect spermatozoa in the female reproductive tract. By means of affinity chromatography the heparin- and Zn2+-binding proteins were isolated from boar seminal plasma and both preparations were analyzed by reverse HPLC. Most of the proteins bound to heparine and Zn2+-ions were classified as spermadhesins. Three fractions binding exclusively Zn2+ were isolated. They differ in amino-acid composition, content of glucosamine and content of protein components revealed by SDS/PAGE.

Laminin interactions with ductal pancreatic adenocarcinoma cells: identification of laminin- and collagen-binding proteins

1990 ◽  
Vol 95 (1) ◽  
pp. 65-74
Author(s):  
M. Mai ◽  
K. Brune ◽  
B. Jacoby ◽  
H.F. Kern ◽  
J. Mollenhauer
Keyword(s):  
Plasma Membrane ◽  
Affinity Chromatography ◽  
Pancreatic Adenocarcinoma ◽  
Binding Proteins ◽  
Collagen Type ◽  
Collagen Type I ◽  
Regulatory Function ◽  
Molecular Heterogeneity ◽  
Type I ◽  
Adenocarcinoma Cells

Laminin promotes the modulation of human pancreatic adenocarcinoma cells from a proliferative to a resting phenotype. This process includes restoration of cell polarity, increase of protein biosynthesis, and increase of glycoprotein secretion. The growth correlates with the amount of laminin coated on the culture dish. Adenocarcinoma cells do not synthesize collagen type I, fibronectin or laminin. Prolonged propagation of the cells on laminin substratum enhances the expression of laminin-binding sites on the cell surface. Laminin binds to cell plasma membrane vesicles with a KB of about 2.2 × 10(10). By affinity chromatography of [35S]methionine-labeled, detergent-extracted, cells on immobilized laminin, a Mr 82,000 polypeptide could be enriched. By simultaneous chromatography on immobilized collagen type I, a Mr 34,000 polypeptide was retained. In laminin overlay experiments on transblotted plasma membrane proteins, Mr 82,000 and 70,000 polypeptides were labeled. Affinity chromatography on laminin-Sepharose of tumor cell membranes from cells grown in nude mice tumors retained Mr 100,000, 82,000, 70,000 and 55,000 polypeptides bound to the column. Endoglycosidase F treatment of these proteins reduced the number of higher Mr proteins, leaving a Mr 70,000 polypeptide, together with smaller peptides. Using the enriched binding protein fraction as antigens, monoclonal antibodies (mAbs) were prepared in mice. Two of the mAbs were further analysed; they recognized simultaneously the complete pattern of high and low Mr polypeptides identified to date by the other methods. Antibody 2A1-H7 was capable of inhibiting attachment and spreading of the cells on laminin and collagen I, but not on tissue-culture plastic. These data may indicate a molecular heterogeneity, partly based on diverse glycosylation, responsible for the variations in the molecular weight of the binding proteins. The adaptation processes of the tumor cells during growth on the extracellular matrix may indicate a regulatory function on tumor growth and metastasis in vivo.

scholarly journals Identification of albumin-binding proteins in capillary endothelial cells.

1988 ◽  
Vol 107 (1) ◽  
pp. 231-239 ◽  
Author(s):  
N Ghinea ◽  
A Fixman ◽  
D Alexandru ◽  
D Popov ◽  
M Hasu ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Specific Binding ◽  
Capillary Endothelium ◽  
Albumin Binding ◽  
Albumin Concentration ◽  
Microvascular Endothelium ◽  
Binding Properties ◽  
Specific Binding Sites ◽  
Capillary Endothelial Cells

Isolated fat tissue microvessels and lung, whose capillary endothelia express in situ specific binding sites for albumin, were homogenized and subjected to SDS-gel electrophoresis and electroblotting. The nitrocellulose strips were incubated with either albumin-gold (Alb-Au) and directly visualized, or with [125I]albumin (monomeric or polymeric) and autoradiographed. The extracts of both microvascular endothelium and the lung express albumin-binding proteins (ABPs) represented by two pairs of polypeptides with major components of molecular mass 31 and 18 kD. The ABP peptides have pIs 8.05 to 8.75. Rabbit aortic endothelium, used as control, does not express detectable amounts of ABPs. The ABPs subjected to electrophoresis bind specifically and with high affinity (Kd = approximately 60 X 10(-9)M) both monomeric and polymeric albumin: the binding is saturable at approximately 80 nM concentration and 50% inhibition is reached at 5.5 micrograms/ml albumin concentration. Sulfhydryl-reducing agents beta-mercaptoethanol and dithiothreitol do not markedly affect the ABPs electrophoretic mobility and binding properties. As indicated by cell surface iodination of isolated capillary endothelium followed by electroblotting, autoradiography, and incubation with Alb-Au, the bands specifically stained by this ligand are also labeled with radioiodine.

3-D organization of fibrinogen receptors using computer reconstruction and whole-mount microscopy

1988 ◽  
Vol 46 ◽  
pp. 30-31
Author(s):  
E. T. O'Toole ◽  
R. R. Hantgan ◽  
J. C. Lewis
Keyword(s):  
Whole Blood ◽  
Colloidal Gold ◽  
Blood Platelets ◽  
Cell Contact ◽  
Computer Assisted ◽  
Adherent Cells ◽  
Differential Centrifugation ◽  
Computer Reconstruction ◽  
Sds Page ◽  
Whole Mount

Thrombocytes (TC), the avian equivalent of blood platelets, support hemostasis by aggregating at sites of injury. Studies in our lab suggested that fibrinogen (fib) is a requisite cofactor for TC aggregation but operates by an undefined mechanism. To study the interaction of fib with TC and to identify fib receptors on cells, fib was purified from pigeon plasma, conjugated to colloidal gold and used both to facilitate aggregation and as a receptor probe. Described is the application of computer assisted reconstruction and stereo whole mount microscopy to visualize the 3-D organization of fib receptors at sites of cell contact in TC aggregates and on adherent cells.Pigeon TC were obtained from citrated whole blood by differential centrifugation, washed with Ca++ free Hank's balanced salts containing 0.3% EDTA (pH 6.5) and resuspended in Ca++ free Hank's. Pigeon fib was isolated by precipitation with PEG-1000 and the purity assessed by SDS-PAGE. Fib was conjugated to 25nm colloidal gold by vortexing and the conjugates used as the ligand to identify fib receptors.

Plasminogen-Plasmin System IX. Specific Binding of Tranexamic Acid to Plasmin

1975 ◽  
Vol 33 (03) ◽  
pp. 573-585 ◽  
Author(s):  
Masahiro Iwamoto
Keyword(s):  
Tranexamic Acid ◽  
Affinity Chromatography ◽  
Dissociation Constant ◽  
Factor Xiii ◽  
Specific Binding ◽  
The Other ◽  
Inhibitory Mechanism ◽  
Binding Studies ◽  
Similar Affinity ◽  
Fibrin Structure

SummaryInteractions between tranexamic acid and protein were studied in respect of the antifibrinolytic actions of tranexamic acid. Tranexamic acid did neither show any interaction with fibrinogen or fibrin, nor was incorporated into cross-linked fibrin structure by the action of factor XIII. On the other hand, tranexamic acid bound to human plasmin with a dissociation constant of 3.5 × 10−5 M, which was very close to the inhibition constant (3.6 × 10−5 M) for this compound in inhibiting plasmin-induced fibrinolysis. The binding site of tranexamic acid on plasmin was not the catalytic site of plasmin, because TLCK-blocked plasmin also showed a similar affinity to tranexamic acid (the dissociation constant, 2.9–4.8 × 10−5 M).In the binding studies with the highly purified plasminogen and TLCK-plasmin preparations which were obtained by affinity chromatography on lysine-substituted Sepharose, the molar binding ratio was shown to be 1.5–1.6 moles tranexamic acid per one mole protein.On the basis of these and other findings, a model for the inhibitory mechanism of tranexamic acid is presented.

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