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.

THYROXINE BINDING PROTEINS OF MOUSE SERUM AT PHYSIOLOGICAL pH

1969 ◽  
Vol 62 (2) ◽  
pp. 234-241
Author(s):  
Amirav Gordon ◽  
Theodore Coutsoftides
Keyword(s):  
Gel Electrophoresis ◽  
Binding Proteins ◽  
Binding Capacity ◽  
Mouse Serum ◽  
Binding Properties ◽  
Agar Gel ◽  
Thyroxine Binding Globulin ◽  
Agar Gel Electrophoresis ◽  
Fractional Distribution

ABSTRACT The binding properties of mouse serum for L-3'5'3,5 tetraiodothyronine (T4) and L-3'3,5, triiodothyronine (T3) were investigated by agar gel electrophoresis in several buffer systems at pH 7.4 and pH 8.6. Mouse serum binds T4 and T3 in the albumin and the α-globulin regions; these were designated as mouse thyroxine binding albumin (TBA) and mouse thyroxine binding globulin (TBG). The fractional distribution of T4 varied with the buffer, the pH and the molarity of the system. Mouse TBG contained two binders, the first with a binding capacity of 14.0 × 10−7 m and the second with a binding capacity of 54.3 × 10−7 m. The effect of various inhibitors on thyroxine binding by mouse TBG suggests that mouse TBG behaves in some respects like human TBG and in other respects like human thyroxine-binding pre-albumin (TBPA).

Binding proteins from fish sera and intrinsic factor compared in vitamin B12 radioassay.

1977 ◽  
Vol 23 (11) ◽  
pp. 2043-2047 ◽  
Author(s):  
D S Ithakissios ◽  
D O Kubiatowicz ◽  
D C Windorski ◽  
J H Wicks
Keyword(s):  
Ionic Strength ◽  
Human Serum ◽  
Chinook Salmon ◽  
Binding Proteins ◽  
Coho Salmon ◽  
Serum Proteins ◽  
Binding Capacity ◽  
Intrinsic Factor ◽  
Serum Samples ◽  
Binding Characteristics

Abstract We compare serum proteins from rainbow trout, chinook salmon, coho salmon, and oyster toadfish with intrinsic factor as binding proteins in a simplified radioassay for B12. Regression analysis of B12 values, determined in 21 serum samples, shows good correlation (r greater than .975) between results for the fish sera and intrinsic factor. The accuracy of the five assays, as evaluated by analytical recovery of B12 added to pooled human serum, ranges from 90 to 110%. Intra-assay precision ranges from 2.6% for coho salmon serum to 5.5% for intrinsic factor, Ionic strength and variations in pH influence binding of [57Co]vit B12 to the fish sera. Maximum binding occurs from pH 6 to 10 at an ionic strength of 0.1 for all sera. The sera are stable for longer than two years when stored at -20 degrees C. Important advantages of fish sera are their high binding capacity (typical assay dilutions range from 1500-fold for trout serum to more than 50 000-fold for chinook salmon); high affinity for B12 (K greater than 10(12) liter/mol); their relative constant binding characteristics as compared to commercial intrinsic factor preparations; and the finding that the accuracy of radioassays with use of fish sera is not significantly affected by the amount of B12 or human serum proteins present.

scholarly journals Delineation of Species-Specific Binding Properties of the CspZ Protein (BBH06) of Lyme Disease Spirochetes: Evidence for New Contributions to the Pathogenesis of Borrelia spp.

2007 ◽  
Vol 75 (11) ◽  
pp. 5272-5281 ◽  
Author(s):  
Elizabeth A. Rogers ◽  
Richard T. Marconi
Keyword(s):  
Lyme Disease ◽  
Ligand Binding ◽  
Dna Sequence ◽  
Molecular Basis ◽  
Serum Proteins ◽  
Specific Binding ◽  
Coiled Coil ◽  
Factor H ◽  
Site Directed Mutagenesis ◽  
Binding Properties

ABSTRACT Borrelia burgdorferi CspZ (TIGR open reading frame designation, BBH06) is part of a functionally related group of proteins that bind one or more members of the factor H (FH) protein family. In this report we assess the conservation, distribution, properties, and ligand binding abilities of CspZ from the three main Borrelia species associated with Lyme disease infections in humans. CspZ (also referred to as BbCRASP-2 in the literature) was found to be highly conserved at the intraspecies level but divergent at the interspecies level. All CspZ orthologs that originated from B. burgdorferi isolates bound FH from a diverse group of mammals. In contrast, CspZ derived from B. garinii and B. afzelii did not. Regardless of the Borrelia species of origin, all CspZ proteins tested bound to unknown ∼60-kDa serum proteins produced by different mammals. To further define the molecular basis for the differential binding of CspZ orthologs to host proteins, DNA sequence, truncation, and site-directed mutagenesis analyses were performed. DNA sequence analyses revealed that B. garinii and B. afzelii CspZ orthologs possess a 64-amino-acid N-terminal domain that is absent from B. burgdorferi CspZ. However, binding analyses of recombinant proteins revealed that this domain does not in and of itself influence ligand binding properties. Truncation and mutagenesis analyses further revealed that the key determinants required for ligand binding are discontinuous and that the presentation of the ligand binding pocket is dependent on alpha helices with high coiled-coil formation probability. The data presented here provide insight into the molecular basis of CspZ-ligand interactions and suggest that CspZ orthologs from diverse Borrelia species can contribute to the host-pathogen interaction through their interaction with serum proteins.

scholarly journals Identification of Novel High Molecular Weight Insulin-Like Growth Factor-Binding Protein-3 Association Proteins in Human Serum1

1998 ◽  
Vol 83 (8) ◽  
pp. 2843-2848
Author(s):  
Paulo F. Collett-Solberg ◽  
Steven E. Nunn ◽  
Tara Beers Gibson ◽  
Pinchas Cohen
Keyword(s):  
Growth Factor ◽  
Human Serum ◽  
Serum Proteins ◽  
Binding Capacity ◽  
Specific Binding ◽  
Insulin Like Growth Factor ◽  
Igf Binding Proteins ◽  
High Affinity ◽  
Acid Labile ◽  
Igfbp 3

abstract The insulin-like growth factor (IGF)-binding proteins (IGFBPs) carry IGFs in serum and regulate their activity and bioavailability. The main IGFBP in serum, IGFBP-3, is known to form a 150-kDa complex with IGFs and the acid-labile subunit (ALS). We investigated the binding of IGFBP-3 to additional association proteins in human serum (IGFBP-3 APs). Ligand blots, column chromatography, and affinity cross-linking experiments revealed the specific binding of IGFBP-3 to at least three novel serum proteins. These techniques demonstrated the presence of proteins with molecular masses of 70, 100, and 150 kDa that bind IGFBP-3 with high affinity. Serum ALS migrated separately (at 88 kDa) from the novel IGFBP-3 APs (as evident by Western immunoblot), and bound IGFBP-3 weakly (by reverse ligand blots). We also demonstrated that large amounts of one of the IGFBP-3 APs and small amounts of ALS were coimmunoprecipitated with IGFBP-3 from human serum. Similar to ALS, these IGFBP-3 APs are acid labile and lose their IGFBP-3 binding capacity after exposure to low pH. We conclude that there are several serum proteins in addition to ALS and IGFs that bind IGFBP-3 with high affinity. These IGFBP-3 APs may serve as an additional reservoir of IGFBP-3 or modulate its functions.

FACTORS INFLUENCING TRIIODOTHYRONINE BINDING PROPERTIES OF LIVER NUCLEAR RECEPTORS

1976 ◽  
Vol 83 (2) ◽  
pp. 293-304 ◽  
Author(s):  
Leslie J. Degroot ◽  
Janine Torresani ◽  
Pierre Carrayon ◽  
Alain Tirard
Keyword(s):  
Nuclear Receptor ◽  
Liver Cell ◽  
Serum Proteins ◽  
Binding Capacity ◽  
Protein S ◽  
Receptor Protein ◽  
Cell Nuclei ◽  
Rat Serum

ABSTRACT Triiodothyronine (T3) may bind directly to receptors present in liver cell nuclei, or may be transported into nuclei by receptor protein(s) present in the cytosol. To evaluate these possibilities, T3 binding was studied in vitro using liver cell nuclei isolated from rats exposed in vivo to very low (H), normal (N), or high levels of T3 (H + T3), and using nuclei incubated in vitro with added cytosol proteins. Ka for T3 was 0.075 ± 0.05 × 1010 m−1 in N, 0.1+0.04 in H, and 0.094 + 0.04 in H + T3, and pg T3 bound/100 μg DNA were 47 ± 17, 31 ± 14, and 29 ± 8 in the three groups. The data indicate no difference in binding capacity between the groups related to prior in vivo exposure to T3, and that T3 may bind directly to empty nuclear receptor sites. Rat liver cytosol proteins added to the in vitro incubation medium always depressed T3 uptake by nuclei. Bovine serum albumin had a similar effect. Large amounts of rat serum proteins depressed uptake, but low levels augmented T3 binding through an unknown mechanism. It is probable that free T3 in serum is in equilibrium with free T3 in the cytosol and nucleus, and binds directly to nuclear receptor proteins without mediation by a cytosol receptor protein.

EFFECT OF pH ON THE BINDING OF THYROXINE TO SERUM PROTEINS

1970 ◽  
Vol 65 (3) ◽  
pp. 409-422 ◽  
Author(s):  
Theodore Coutsoftides ◽  
Amirav Gordon
Keyword(s):  
Serum Albumin ◽  
Serum Proteins ◽  
Binding Capacity ◽  
Ph Sensitive ◽  
The Other ◽  
Rat Serum ◽  
Effect Of Ph ◽  
Thyroxine Binding Globulin ◽  
Other Hand ◽  
Maximal Binding

ABSTRACT The maximal binding capacity of human thyroxine-binding globulin (TBG) was found to decrease, and that of thyroxine-binding pre-albumin (TBPA) to increase with an increase in pH in the range of pH 7.2 to 8.2. On the other hand, the transfer of T4125I from the serum to a weak binder (Sephadex) was found to decrease with increasing pH. The same phenomena was shown to exist in mouse and rat serum, and to be blocked by DNP, a potent TBA & TBPA binding inhibitor. It is suggested that serum albumin may play a pH sensitive role in the T4 transfer to tissue.

scholarly journals Changing pattern of cyclic AMP-binding proteins during germination of Mucor racemosus sporangiospores

1979 ◽  
Vol 182 (2) ◽  
pp. 547-554 ◽  
Author(s):  
M Orlowski
Keyword(s):  
Cyclic Amp ◽  
Time Course ◽  
Binding Proteins ◽  
Dissociation Constants ◽  
Binding Capacity ◽  
Specific Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mucor Racemosus ◽  
Response Curves ◽  
Kd Values

Interation of cyclic AMP with a profoundly changing pattern of specific binding proteins was shown during aerobic germination of sporangiospores from the fungus Mucor racemosus. 32P-labeled 8-azido-cycli AMP, an analogue of cyclic AMP that forms a covalent linkage with the binding proteins under u.v. light, was used as the ligand. Binding proteins carrying this photoaffinity label were separated by polyacrylamide-gel electrophoresis and identified by radioautography. Equibiltrium dissociation constants (Kd) and binding-response curves in the presence of competing nucleotides were identical for both 8-azido-cyclic [32P]AMP and cyclic [3H]AMP. A quantitative binding assay with both 8-azido-cyclic [32P]AMP and cyclic [3H]AMP over the time course of sporangiospore germination indicated a parallel relationship between cyclic AMP-binding capacity and the intracellular concentrations of cyclic AMP reported in a previous study [Paznokas & Sypherd (1975) J. Bacteriol. 124, 134–139]. Both of these parameters attained transient high values at a time of development when addition of exogenous cyclic AMP prevents hyphal-germ-tube emergence. The measured Kd values did not change during sport germination.

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.

Plasma proteins as biomarkers of the aging process

1995 ◽  
Vol 268 (2) ◽  
pp. R536-R548 ◽  
Author(s):  
R. Vranckx ◽  
L. Savu ◽  
N. Lambert ◽  
G. V. de Conchard ◽  
R. Grosse ◽  
...  
Keyword(s):  
Aging Process ◽  
Serum Proteins ◽  
Enzyme Inhibitor ◽  
Binding Capacity ◽  
Protein Biosynthesis ◽  
Exclusion Criterion ◽  
Protein Patterns ◽  
Rat Serum ◽  
Crossed Immunoelectrophoresis ◽  
Age Related

This study was designed to characterize the rat serum proteins as biomarkers of the normal aging process. Crossed immunoelectrophoresis or electroimmunodiffusion quantitation of proteins was performed in rats aged 6, 12, 24, and 30 mo. Selection of healthy animals was based on confrontation of crossed immunoelectrophoresis patterns with those of experimentally inflamed young adults and with individual anatomopathological data. Convergence of inflammatory patterns and severe histological lesions was the exclusion criterion. Senescence-induced decrease was demonstrated for eight proteins [negative senescence reactants (SRs-)] and increase for six proteins [positive SRs (SRs+)]. Most SRs belonged to the class of proteins responsive to acute inflammation [acute phase reactants (APRs)]. One SR+, the thyroxine-binding globulin, a high-affinity thyroid hormone binder, emerged as a particularly reliable senescence biomarker, showing the highest aging-related variation (8-fold increase from 6 to 30 mo) and not belonging to the APR class. Chronic treatment with perindopril, an angiotensin I-converting enzyme inhibitor used in heart and renal disease therapy, significantly enhanced thyroxine-binding capacity, possibly by preventing age-related alterations of serum lipids. Serum protein patterns prove valuable both as indexes for selecting aging animals free from superimposed pathologies and as parameters of senescence-induced changes in protein biosynthesis.

scholarly journals Beyond antibodies ? lessons from bacterial ?immunity?

2006 ◽  
Vol 27 (2) ◽  
pp. 80
Author(s):  
Stewart D Nuttall ◽  
Suzy M Juraja ◽  
Jennifer A Carmichael
Keyword(s):  
Binding Proteins ◽  
Specific Binding ◽  
Immune Surveillance ◽  
Specific Protein ◽  
Protein Scaffolds ◽  
Immune Systems ◽  
Alternative Protein ◽  
Human Proteins ◽  
Specific Binding Molecules ◽  
Highly Evolved

Isolation and production of highly specific protein-based binding molecules are crucial to the ever expanding diagnostics, therapeutics and protein array fields. Traditionally, such reagents have been sourced from vertebrate immune systems, where antibodies have evolved over millennia into highly effective molecules of immune surveillance capable of targeting a huge range of targets in response to infection and disease. Now, a growing number of alternative protein scaffolds are being investigated as specific binding molecules incorporating a diverse and powerful range of binding and recognition interfaces. These are being sourced from human proteins, from alternative immune molecules present in evolutionarily old vertebrates, and from highly evolved binding proteins in prokaryotic systems.

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