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At least 19 recordsLinked to original sources
PubMed1979

Competitive binding studies with glucocorticoid receptors from rat-thymus cells: differential temperature-dependence of steroid binding.

Competitive steroid-binding studies were performed with intact rat thymus cells and with cytosol preparations at different temperatures using [1,2-3H]dexamethasone as the labelled ligand. Steroids lacking a 17 alpha-hydroxyl group, such as corticosterone, were better able to compete with [1,2-3H]dexamethasone for binding to glucocorticoid receptors at 0 degrees C than compounds containing a 17 alpha-hydroxyl substituent, such as cortisol. At 37 degrees C the reverse was true. This temperature-dependent change in relative affinities appeared to be unrelated to steroid metabolism or receptor activation, and to depend only on the thermodynamic parameters of the steroid--receptor interaction. Relative biological activities for different steroids agree more closely with the relative affinities determined at 37 degrees C than with those determined at lower temperatures.

Animals↗
PubMed1975

The thyroxine-binding properties of serum proteins. A competitive binding technique employing sephadex G-25.

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 9.5x10(9), 1.6x10(8) and 3.1x10(5) 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 8.9x10(9), 1-4 X 10(8) and 3.5x10(5) 1/mol. Human TBG had a mean binding capacity of 21.3 mug/100 ml and that of ovie TBG was 12.8 mug/100 ml. The other specific binding proteins (TBPA in man and TBP-2 in sheep) had mean binding capacities of 307 and 359 mug/100 ml respectively. Two functionally different T4-binding proteins were identified in rat serum.

Animals↗
PubMed1992

Characterization of carbohydrate-binding specificity of concanavalin A by competitive binding of pyridylamino sugar chains.

Carbohydrate-binding specificity of Con A was characterized by competitive binding studies of pyridylamino (PA) sugar chains. PA-derivatives of 17 oligomannose-type sugar chains, Man1-9GlcNAc2-PA, and those of three complex-type sugar chains were used as ligands. The ratios of bound and free sugar concentrations, [LS]/[S], were determined by means of microequilibrium dialysis followed by high performance liquid chromatography as already reported [Mega, T. & Hase, S. (1991) J. Biochem. 109, 600-603]. The association constant, Ka, was calculated from [LS]/[S] of a sugar chain and that of a standard sugar chain by using the equation Ka = Ka0 x ([S0]/[LS0]) x ([LS]/[S]), where Ka0, [S0], and [LS0] are the association constant, and the free and bound ligand concentrations of the standard sugar chain, respectively. This calculation was effective for the determination of Ka of ligands with similar affinities to the standard sugar chain. The carbohydrate structures with highest affinity for Con A among those tested were found to be: [formula: see text]

Amino Sugars↗
PubMed1979

Competitive binding radioassay for 5-fluorodeoxyuridine 5'-monophosphate in tissues.

A competitive binding radioassay has been developed for 5-fluorodeoxyuridine 5'-monophosphate, based on the tight binding of this potent inhibitor to thymidylate synthetase (EC 2.1.1.45). Unbound ligand may be separated from that bound to enzyme by precipitating the intact inhibitor-enzyme complex with trichloroacetic acid. Scatchard plot analysis using a two-site model for binding yielded apparent dissociation constants of 1.2 x 10(-11) and 1.7 x 10(-10) M from a least-squares computer fit of the data. 5-Fluorodeoxyuridine 5'-monophosphate could be detected in the range of 0.02 to 2.0 pmol with no apparent interference by other substances. Assay of 5-fluorodeoxyuridine 5'-monophosphate levels in L1210 ascites tumor following 5-fluorouracil in vivo revealed peak levels occurring within the first hr with a subsequent disappearance half-life of 3.9 hr. Close agreement was found between the previously described enzyme inhibition assay and the more rapid and sensitive competitive binding method.

Animals↗
PubMed1977

An automated competitive binding procedure for measuring thyroxine in serum.

A competitive binding assay for serum thyroxine has been automated, with the use of small, reusable Sephadex columns to separate thyroxine from endogenous thyroid-binding globulin and later to separate the bound and free thyroxine. Sixty samples an hour are run through columns, which are held in an aluminum turntable rotated by a fraction collector motor. Reagents and samples are fed to the columns by a proportioning pump. Waste eluates are collected and drained to the sink by a Teflon tray positioned between the columns and counting tubes, also held by the turntable. A cut out area in the tray allows one to collect the bound fraction, which can then be counted in a scintillation counter. Values are calculated and printed by a desk calculator interfaced with the scintillation counter. The "day-to-day" CV for this method is 5%. The accuracy is satisfactory when tested by comparison with other methods, by recoveries, and by linearity of dilutions.

Autoanalysis↗
PubMed1978

Immunochemical studies on blood groups LXVI. Competitive binding assays of A1 and A2 blood group substances with insolubilized anti-A serum and insolubilized A agglutinin from Dolichos biflorus.

Competitive binding assays using 3H-labeled blood group A substance and insolubilized Dolichos biflorus lectin or human anti-A were carried out, measuring competition by blood group A1 and A2 glycoproteins, and by unabsorbed anti-A sera, and with these sera absorbed with the A1 and A2 glycoproteins. With Dolichos lectin specific for (formula: see text) A1 substances had about 11 times as many determinants as did A2 substances, but the slopes of the lines in the competitive binding assays were the same. With insolubilized anti-A, A2 substances gave lines of lower slopes. Although individual A1 populations varied in the amounts giving 50% inhibition in the assays, as did A2 substances, the slopes of the lines for the A1 substances were the same and always higher than the slopes of the lines for the A2 substances. Competitive binding assays with unabsorbed anti-A sera and with these sera absorbed with insoluble polyleucyl A1 and A2 substances showed that partial absorption of polyleucyl A1 substances left antibodies of lower slope in the supernate, whereas absorption with polyleucyl A2 substance left antibodies (anti-A1) having the same or an even higher slope than the unabsorbed sera. The findings indicate that human A1 and A2 glycoproteins differ in their determinants, and that A2 specificity is determined by the type 2 chain in which the A trisaccharide (formula: see text) is linked beta 1 leads to 4 to DGlcNAc, whereas the A1 specificity is determined by the type 1 chain in which this trisaccharide is linked beta 1 leads to 3 to DGlcNAc; most of the determinants in the glycoproteins have a second LFuc linked alpha 1 leads to 3 and alpha 1 leads to 4 to the DGlcNAc of the type 2 and type 1 chains, respectively.

ABO Blood-Group System↗
PubMed1975

Competitive binding activity of angiotensin II analogues in an adrenal cortex radioligand-receptor assay;.

The competitive binding activities of a number of angiotensin analogues were determined in a radioligand-receptor assay employing bovine adrenal crotex homogenate and [125I] iodoangiotensin II. This assay system has been shown to provide a precise and convenient method for evaluation and comparison of the binding-inhibition potencies of angiotensin II derivatives with agonist and antagonist activities. Agonist analogues of angiotensin II showed competitive binding activities in proportion to their known biological activities upon aldosterone production by the adrenal zona glomerulosa. Thus, the [Des-Asp1] heptapeptide of angiotensin II was equipotent with the native octapeptide in terms of binding-inhibition activity, and the [Sar1] derivative of angiotensin II was more potent than the native peptide. By contrast, the [Des-Asp1, Des-Arg2] hexapeptide and the [Des-Phe8] heptapeptide showed less than 1% of the activity of angiotensin II. Angiotensin II antagonists formed by C-terminal substitution of the octapeptide with isoleucine or alanine were also found to exhibit binding-inhibition activities in proportion to their known potencies as antagonists of the action of angiotensin II upon smooth muscle. Certain antagonists, such as [1-guanidoacetic, 8-isoleucine]-angiotensin II and [1-sarcosine, 8-isoleucine]-angiotensin II displayed significantly greater binding-inhibition potency than E1Asp1, Ileu5,-angiotensin II in the adrenal receptor system. Several, such as [Sar1, Ala8]-angiotensin II and [MeAla1, Ileu8]-antiotensin II were approximately equipotent with angiotensin II, while others such as [1-succinic acid, 8-alanine]-angiotensin II and [D-Ileu8]-angiotensin II showed no significant binding-inhibition activity. The relative binding-inhibition potencies of the antagonist peptides showed a general correlation with the pA2 values of the same components determined upon the smooth muscle response of the isolated aortic strip. The binding-inhibition potency of angiotensin II antagonists was also strongly influenced by the charge of the N-terminal residue, with enhancement of activity by more basic substituents and abolition of activity by highly acidic residues. The influence of the basicity of the N-terminus upon receptor binding was also observed with the agonist analogue [Sar1]-angiotensin II, which showed a 2- to 3-fold increase in binding-inhibition potency in comparison to native angiotensin II. The significant enhancement of binding-inhibition potency by N-terminal sarcosine substitution is attributable to higher affinity of the modified peptide for the angiotensin II receptor site, and is consistent with the increased activity of [Sar1]-angiotensin II upon smooth muscle and aldosterone production in vitro. The determination of binding-inhibition activity in the adrenal radioligand receptor assay provides a valid and convenient method for analysis of the role of binding affinity in the actions of competitive antagonists upon the responses of target cells to angiotensin II.

Adrenal Cortex↗
PubMed1992

Process monitoring by flow-injection immunoassay. Evaluation of a sequential competitive binding assay.

A new variation on the theme of flow-injection binding assays is presented, namely the sequential competitive binding assay, in which the sample containing the native antigen is first introduced into the flow system followed by a pulse of labelled antigen. The flow-injection binding assay was used in monitoring the effluent from column chromatographic separations of proteins. By applying a computer-based evaluation system, concentrations are automatically read and compensation is made for denaturation in the affinity sorbent.

Binding, Competitive↗
PubMed1978

Evaluation of a competitive binding assay for cortisol using horse transcortin.

A non-chromatographic competitive binding assay (CBA) using horse transcortin has been employed in the routine measurement of cortisol in plasma, urine and amniotic fluids. Comparing the values with those of a radioimmunoassay (RIA) or a fluorimetric method (FM) an excellent correlation between the three methods both in plasma and urine has been calculated in normal subjects and in patients with various endocrine disorders. In amniotic fluids, however, there were discrepancies between CBA and RIA. Whereas CBA showed no differences, RIA gave significantly higher values in amniotic fluids of female than of male fetuses. Elevated free plasma cortisol levels observed in patients with prostatic cancer after diethyl stilboestrol diphosphate therapy did not correlate with unconjugated urinary cortisol concentration as measured with CBA and FM. In newborns, a relatively high plasma level found 12 hours after birth was followed by a nadir on the 2nd and 3rd day of life and by an increase until levels of adults on the 5th day of life were reached.

Amniotic Fluid↗
PubMed1978

Measurement of adenosine 3':5'-cyclic monophosphate by competitive binding to salt-dissociated protein kinase.

An assay for cyclic AMP is described which takes advantage of the high affinity of the dissociated receptor moiety of cyclic AMP-dependent protein kinase I for the nucleotide. The kinase is kept dissociated by salt (800 mM-NaCl/30mM-EDTA). In the presence of a simply prepared heat-stable protein fraction the binding reagent is stable for the time needed to reach equilibrium of binding. A simple procedure [precipitation with poly-(ethylene glycol) followed by DEAE-cellulose chromatography] is described for the separation of protein kinase I from other binding proteins for cyclic AMP in rabbit skeletal muscle. The sensitivity, precision, reproducibility and specificity of the assay compared favourably with those of other cyclic AMP assays. The main advantage of the present assay is its resistance towards non-specific interference from a number of salts, tissue-culture media and substances found in crude tissue extracts. The reliability of cyclic AMP measurement directly in crude tissue extracts was ensured by removal of the assayable cyclic AMP with cyclic nucleotide phosphodiesterase digestion or adsorption with antibody against cyclic AMP, by comparison with measurement in tissue extracts purified by chromatography on QAE-Sephadex or sequentially on Dowex 50, and aluminium oxide as well as by dilution and recovery experiments.

Binding, Competitive↗