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.

scholarly journals Specific binding sites for albumin restricted to plasmalemmal vesicles of continuous capillary endothelium: receptor-mediated transcytosis.

1986 ◽  
Vol 102 (4) ◽  
pp. 1304-1311 ◽  
Author(s):  
L Ghitescu ◽  
A Fixman ◽  
M Simionescu ◽  
N Simionescu
Keyword(s):  
Plasma Membrane ◽  
Binding Sites ◽  
Specific Binding ◽  
Short Exposure ◽  
Gold Complex ◽  
Membrane Microdomains ◽  
Capillary Endothelium ◽  
Plasmalemmal Vesicles ◽  
Specific Binding Sites

The interaction of homologous and heterologous albumin-gold complex (Alb-Au) with capillary endothelium was investigated in the mouse lung, heart, and diaphragm. Perfusion of the tracer in situ for from 3 to 35 min was followed by washing with phosphate-buffered saline, fixation by perfusion, and processing for electron microscopy. From the earliest time examined, one and sometimes two rows of densely packed particles bound to some restricted plasma membrane microdomains that appeared as uncoated pits, and to plasmalemmal vesicles open on the luminal front. Morphometric analysis, using various albumin-gold concentrations, showed that the binding is saturable at a very low concentration of the ligand and short exposure. After 5 min, tracer-carrying vesicles appeared on the abluminal front, discharging their content into the subendothelial space. As a function of tracer concentration 1-10% of plasmalemmal vesicles contained Alb-Au particles in fluid phase; from 5 min on, multivesicular bodies were labeled by the tracer. Plasma membrane, coated pits, and coated vesicles were not significantly marked at any time interval. Heparin or high ionic strength did not displace the bound Alb-Au from vesicle membrane. No binding was obtained when Alb-Au was competed in situ with albumin or was injected in vivo. Gold complexes with fibrinogen, fibronectin, glucose oxidase, or polyethyleneglycol did not give a labeling comparable to that of albumin. These results suggest that on the capillary endothelia examined, the Alb-Au is adsorbed on specific binding sites restricted to uncoated pits and plasmalemmal vesicles. The tracer is transported in transcytotic vesicles across endothelium by receptor-mediated transcytosis, and to a lesser extent is taken up by pinocytotic vesicles. The existence of albumin receptors on these continuous capillary endothelia may provide a specific mechanism for the transport of albumin and other molecules carried by this protein.

Assay performance and tracer properties for two analog-based assays of free triiodothyronine.

1986 ◽  
Vol 32 (3) ◽  
pp. 465-469 ◽  
Author(s):  
T A Wilkins ◽  
J E Midgley ◽  
R A Stevens ◽  
I Caughey ◽  
N Barron
Keyword(s):  
Binding Proteins ◽  
Specific Binding ◽  
Albumin Concentration ◽  
Free Triiodothyronine ◽  
Nonesterified Fatty Acids ◽  
Binding Characteristics ◽  
Specific Binding Sites ◽  
Assay Performance ◽  
Wide Range

Abstract We determined binding characteristics of the triiodothyronine (T3) analog tracer used in the Amerlex and Amerlex-M FT3 radioimmunoassay for the three endogenous binding proteins in serum: thyroxin-binding globulin (TBG), thyroxin binding prealbumin (PA), and albumin. Both T3 and its analog bind to the same sites on TBG and PA. However, the analog has significantly lower association constants (1.0% and 3.8%, respectively, of T3 binding affinity) and it binds to different sites on albumin. Analog binding is characterized by two (weak) specific binding sites [K = 0.46 (SD 0.03) X 10(5) L/mol]; T3 is bound at about 28 very weak, nonspecific sites [K = 0.41 (SD 0.03) X 10(4) L/mol]. Sera from healthy subjects with a wide range of concentrations of binding proteins showed no interference from analog binding in the FT3 assay. In contrast, in vitro studies of albumin binding revealed a weak dependence of both assays on albumin concentration (0.05 pmol of FT3 per gram of albumin per liter), an interference probably unimportant for most laboratory samples. Nonesterified fatty acids (NEFA) and the T3 analog apparently bind to different sites on albumin; thus the Amerlex FT3 assay is insensitive to moderately increased concentrations of NEFA in serum.

Specific albumin binding to microvascular endothelium in culture

1988 ◽  
Vol 254 (3) ◽  
pp. H425-H437 ◽  
Author(s):  
J. E. Schnitzer ◽  
W. W. Carley ◽  
G. E. Palade
Keyword(s):  
Specific Binding ◽  
Molecular Transport ◽  
Cell Number ◽  
Dissociation Rate ◽  
Affinity Constant ◽  
Scatchard Analysis ◽  
Albumin Binding ◽  
Microvascular Endothelium ◽  
Rat Serum ◽  
Rate Analysis

The specific binding of rat serum albumin (RSA) to confluent microvascular endothelial cells in culture derived from the vasculature of the rat epididymal fat pad was studied at 4 degrees C by radioassay and immunocytochemistry. Radioiodinated RSA (125I-RSA) binding to the cells reached equilibrium at approximately 20 min incubation. Albumin binding was a slowly saturating function over concentrations ranging from 0.01 to 50 mg/ml. Specific RSA binding with a moderate apparent affinity constant of 1.0 mg/ml and with a maximum binding concentration of 90 ng/cm2 was immunolocalized with anti-RSA antibody to the outer (free) side of the endothelium. Scatchard analysis of the binding yielded a nonlinear binding curve with a concave-upward shape. Dissociation rate analysis supports negative cooperativity of albumin binding, but multiple binding sites may also be present. Albumin binding fulfilled many requirements for ligand specificity including saturability, reversibility, competibility, and dependence on both cell type and cell number. The results are discussed in terms of past in situ investigations on the localization of albumin binding to vascular endothelium and its effect on transendothelial molecular transport.

scholarly journals Synthesis and application of photoaffinity analogues of inositol 1,4,5-trisphosphate selectively substituted at the 1-phosphate group

1990 ◽  
Vol 272 (3) ◽  
pp. 817-825 ◽  
Author(s):  
R Schäfer ◽  
M Nehls-Sahabandu ◽  
B Grabowsky ◽  
M Dehlinger-Kremer ◽  
I Schulz ◽  
...  
Keyword(s):  
Binding Sites ◽  
Binding Proteins ◽  
Inositol Phosphates ◽  
Specific Binding ◽  
Acinar Cells ◽  
Phosphate Group ◽  
Pancreatic Acinar Cells ◽  
Maximal Effect ◽  
Microsomal Preparation ◽  
Specific Binding Sites

We have synthesized two photolabile arylazido-analogues of Ins(1,4,5)P3 selectively substituted at the 1-phosphate group for determination of Ins(1,4,5)P3-binding proteins. These two photoaffinity derivatives, namely N-(4-azidobenzoyl)aminoethanol-1-phospho-D-myo-inositol 4,5-bisphosphate (AbaIP3) and N-(4-azidosalicyl)aminoethanol-1-phospho-D-myo-inositol 4,5-bisphosphate (AsaIP3), bind to high affinity Ins(1,4,5)P3-specific binding sites at a 9-fold lower affinity (Kd = 66 and 70 nM) than Ins(1,4,5)P3 (Kd = 7.15 nM) in a fraction from rat pancreatic acinar cells enriched in endoplasmic reticulum (ER). Other inositol phosphates tested showed comparable (DL-myo-inositol 1,4,5-trisphosphothioate, Kd = 81 nM) or much lower affinities for the binding sites [Ins(1,3,4,5)P4, Kd = 4 microM; Ins(1,4)P2, Kd = 80 microM]. Binding of AbaIP3 was also tested on a microsomal preparation of rat cerebellum [Kd = 300 nM as compared with Ins(1,4,5)P3, Kd = 45 nM]. Ca2+ release activity of the inositol derivatives was tested with AbaIP3. It induced a rapid and concentration-dependent Ca2+ release from the ER fraction [EC50 (dose producing half-maximal effect) = 3.1 microM] being only 10-fold less potent than Ins(1,4,5)P3 (EC50 = 0.3 microM). From the two radioactive labelled analogues ([3H]AbaIP3 and 125I-AsIP3) synthesized, the radioiodinated derivative was used for photoaffinity labelling. It specifically labelled three proteins with apparent molecular masses of 49, 37 and 31 kDa in the ER-enriched fraction. By subfractionation of this ER-enriched fraction on a Percoll gradient the 37 kDa Ins(1,4,5)P3 binding protein was obtained in a membrane fraction which showed the highest effect in Ins(1,4,5)P3-inducible Ca2+ release (fraction P1). The other two Ins(1,4,5)P3-binding proteins, of 49 and 31 kDa, were obtained in fraction P2, in which Ins(1,4,5)P3-induced Ca2+ release was half of that obtained in fraction P1. We conclude from these data that the 37 kDa and/or the 49 and 31 kDa proteins are involved in Ins(1,4,5)P3-induced Ca2+ release from the ER of rat pancreatic acinar cells.

scholarly journals Changes in IGFs in cardiac tissue following myocardial infarction

1999 ◽  
Vol 163 (3) ◽  
pp. 433-445 ◽  
Author(s):  
KG Matthews ◽  
GP Devlin ◽  
JV Conaglen ◽  
SP Stuart ◽  
W Mervyn Aitken ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Binding Proteins ◽  
Cardiac Tissue ◽  
Apoptotic Cell ◽  
Specific Binding ◽  
Cell Types ◽  
Necrotic Tissue ◽  
Igf I ◽  
The Relationship

We have studied changes in the IGF axis in an ovine model of myocardial infarction (MI), in order to determine the relationship between time-based changes in post-infarct myocardium and IGF levels. IGF localization was studied by immunocytochemistry, production by in situ hybridization, and specific binding by radioligand studies. In surviving tissue, IGF-I peptide localized to cardiomyocytes, with strongest immunostaining at 1 and 2 days post-infarct in the immediate border area adjoining the infarct, where IGF-I mRNA also increased, reaching a maximum at 2 days. Binding of radiolabelled IGF-I in surviving tissue was initially lower than that seen in cardiomyocytes in control myocardium, subsequently increasing to become significantly greater by 6 days post-infarct. In necrotic tissue, IGF-I peptide was still detectable in cardiomyocytes at 0.5 days post-infarct, but had cleared from this area by 1 day, becoming detectable again at 6 days post-infarct in macrophages and fibroblasts infiltrating the repair zone. IGF-I mRNA was not detected in necrotic tissue until 6 days, when probe hybridized to macrophages and fibroblasts. Within the necrotic zone, high levels of radiolabelled IGF-I binding to a combination of receptors and binding proteins were observed in cardiomyocytes in islands of viable tissue located close to the border. Weak immunostaining for IGF-II was observed in cardiomyocytes of the surviving tissue. IGF-II mRNA was not detected in either surviving or necrotic areas. Binding of radiolabelled IGF-II was predominantly to macrophages in both surviving and infarct areas, although as with IGF-I, high levels of binding of radiolabelled IGF-II to a combination of receptors and binding proteins were observed in islands of viable tissue close to the border within the necrotic area. We conclude that, following MI, surviving cardiomyocytes at the infarct border show marked changes in IGF-I localization, production, and specific binding, indicating that the IGF axis is directly involved in post-infarct events, possibly in the maintenance of cardiac function by the induction of hypertrophy and in cell survival by decreasing apoptotic cell death, which has been demonstrated in other cell types.

Studies on Corticosterone–Receptor Complexes from Mouse Placenta

1974 ◽  
Vol 52 (3) ◽  
pp. 190-195 ◽  
Author(s):  
Ming D. Wong ◽  
A. F. Burton
Keyword(s):  
Binding Sites ◽  
Time Course ◽  
Specific Binding ◽  
Receptor Site ◽  
Binding Properties ◽  
Receptor Complexes ◽  
Specific Binding Sites ◽  
Mouse Placenta ◽  
Vitro Binding

The in vitro binding of radioactive steroids to components of mouse placental nuclei and cytoplasm was investigated using Sephadex or charcoal to remove unbound steroid. Specificity was indicated in competition experiments using excess unlabelled competing steroids. Only the active glucocorticoids formed complexes that could be isolated from the nucleus. The binding properties of the cytoplasmic steroid–receptor complex were studied. From the time course of binding the complex was shown to be more stable at 0° than at 37°, and the distribution of receptors in the cytosol appeared to be homogeneous. The complex was labile to heat and to proteolytic digestion but did not appear to be affected by nucleases or sulfhydryl reagents. Kinetic analysis revealed the presence of high affinity specific binding sites with a dissociation constant of 17.5 nM and a receptor site concentration of 0.26 pmol/mg protein. The corticosterone isolated from nuclear complexes and dexamethasone from cytoplasmic complexes were identified by chromatography and by cocrystallization as the unchanged steroid in each case.

THE THYROXINE-BINDING PROPERTIES OF SERUM PROTEINS. A COMPETITIVE BINDING TECHNIQUE EMPLOYING SEPHADEX G-25

1975 ◽  
Vol 65 (3) ◽  
pp. 319-332 ◽  
Author(s):  
R. L. SUTHERLAND ◽  
M. W. SIMPSON-MORGAN
Keyword(s):  
Binding Proteins ◽  
Serum Proteins ◽  
Binding Capacity ◽  
Specific Binding ◽  
Competitive Binding ◽  
Association Constants ◽  
Binding Properties ◽  
Rat Serum ◽  
Human Proteins ◽  
Dilute Blood

SUMMARY A competitive binding technique is described for the estimation of the thyroxine (T4)-binding properties of serum proteins in dilute blood serum and lymph. When used in conjunction with an assay for total T4 the following parameters can be estimated: the number of functionally different T4 binding proteins, their individual association constants and binding capacities for T4, the amount of T4 which is bound to each binding species, and the concentration of unbound (free) T4. Both human and sheep serum have three functionally different T4-binding proteins. The association constants for the three human proteins were 9·5 × 109, 1·6 × 108 and 3·1 × 105 1/mol for T4-binding globulin (TBG), T4-binding prealbumin (TBPA) and serum albumin, respectively. The corresponding sheep proteins, TBG, TBP-2 and albumin, had association constants of 8·9 × 109, 1·4 × 108 and 3·5 × 1051/mol. Human TBG had a mean binding capacity of 21·3 μg/100 ml and that of ovine TBG was 12·8 μg/100 ml. The other specific binding proteins (TBPA in man and TBP-2 in sheep) had mean binding capacities of 307 and 359 μg/100 ml respectively. Two functionally different T4-binding proteins were identified in rat serum.

scholarly journals Direct in vivo demonstration by radioautography of specific binding sites for calcitonin in skeletal and renal tissues of the rat.

1980 ◽  
Vol 85 (3) ◽  
pp. 682-694 ◽  
Author(s):  
H Warshawsky ◽  
D Goltzman ◽  
M F Rouleau ◽  
J J Bergeron
Keyword(s):  
Binding Sites ◽  
Alveolar Bone ◽  
Specific Binding ◽  
Biologically Active ◽  
Target Cells ◽  
Rat Tissues ◽  
Specific Binding Sites ◽  
Calcitonin Receptors

An in vivo binding assay using radioautography was employed to visualize calcitonin receptors in rat tissues. At 2 min after intravenous injection of biologically active 125I-salmon calcitonin, free hormone was separated from bound hormone by intracardiac perfusion with lactated Ringer's followed by fixation with 2.5% glutaraldehyde. Various tissues were removed and processed for light and electron microscope radioautography. These were compared to tissues removed from animals that received identical amounts of labeled hormone with a large excess of unlabeled calcitonin. Among the tissues investigated, kidney and bone demonstrated labeling. In kidney, most silver grains were located over vesicles below the brush border of cells of theproximal convoluted tubules. These grains were still present after simultaneous injection of excess unlabeled hormone and most likely represented binding to sites involved with ingestion and degradation of hormone from the urinary filtrate. In contrast, grains localized to the basal surfaces of distal convoluted tubule cells were significantly reduced in number in control animals and represented sites of saturable, specific hormone binding. In bone, specific binding sites were found only at the periphery of osteoclasts. These labeled cells were located at resorption sites examined in tibia, humerus, and alveolar bone. This demonstration of the localization of 124I-calcitonin in situ provides a new approach for study the interaction of calcium-regulating hormones with their target cells.

Quantitative analysis of albumin uptake and transport in the rat microvessel endothelial monolayer

2003 ◽  
Vol 284 (1) ◽  
pp. L187-L196 ◽  
Author(s):  
Theresa A. John ◽  
Stephen M. Vogel ◽  
Chinnaswamy Tiruppathi ◽  
Asrar B. Malik ◽  
Richard D. Minshall
Keyword(s):  
Specific Binding ◽  
Fluid Phase ◽  
Scatchard Analysis ◽  
Albumin Binding ◽  
Endothelial Monolayer ◽  
Site Model ◽  
Specific Binding Sites ◽  
High K ◽  
Secondary Antibodies ◽  
Uptake And Transport

We determined the concentration dependence of albumin binding, uptake, and transport in confluent monolayers of cultured rat lung microvascular endothelial cells (RLMVEC). Transport of 125I-albumin in RLMVEC monolayers occurred at a rate of 7.2 fmol · min−1 · 106cells−1. Albumin transport was inhibited by cell surface depletion of the 60-kDa albumin-binding glycoprotein gp60 and by disruption of caveolae using methyl-β-cyclodextrin. By contrast, gp60 activation (by means of gp60 cross-linking using primary and secondary antibodies) increased 125I-albumin uptake 2.3-fold. At 37°C, 125I-albumin uptake had a half time of 10 min and was competitively inhibited by unlabeled albumin (IC50= 1 μM). Using a two-site model, we estimated by Scatchard analysis the affinity ( K D) and maximal capacity (Bmax) of albumin uptake to be 0.87 μM ( K D1) and 0.47 pmol/106 cells (Bmax1) and 93.3 μM ( K D2) and 20.2 pmol/106 cells (Bmax2). At 4°C, we also observed two populations of specific binding sites, with high ( K D1 = 13.5 nM, 1% of the total) and low ( K D2 = 1.6 μM) affinity. On the basis of these data, we propose a model in which the two binding affinities represent the clustered and unclustered gp60 forms. The model predicts that fluid phase albumin in caveolae accounts for the bulk of albumin internalized and transported in the endothelial monolayer.

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