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PubMed1998

Competitive calcium binding: implications for dendritic calcium signaling.

Action potentials evoke calcium transients in dendrites of neocortical pyramidal neurons with time constants of < 100 ms at physiological temperature. This time period may not be sufficient for inflowing calcium ions to equilibrate with all present Ca2+-binding molecules. We therefore explored nonequilibrium dynamics of Ca2+ binding to numerous Ca2+ reaction partners within a dendritelike compartment using numerical simulations. After a brief Ca2+ influx, the reaction partner with the fastest Ca2+ binding kinetics initially binds more Ca2+ than predicted from chemical equilibrium, while companion reaction partners bind less. This difference is consolidated and may result in bypassing of slow reaction partners if a Ca2+ clearance mechanism is active. On the other hand, slower reaction partners effectively bind Ca2+ during repetitive calcium current pulses or during slower Ca2+ influx. Nonequilibrium Ca2+ distribution can further be enhanced through strategic placement of the reaction partners within the compartment. Using the Ca2+ buffer EGTA as a competitor of fluo-3, we demonstrate competitive Ca2+ binding within dendrites experimentally. Nonequilibrium calcium dynamics is proposed as a potential mechanism for differential and conditional activation of intradendritic targets.

Action Potentials↗
PubMed1984

A competitive ligand binding assay for measurement of thyroid hormone-binding inhibitor in serum and tissues.

A competitive ligand-binding assay (CLBA) is described for measurement of an inhibitor(s) of serum binding of T4 in ether extracts of serum and in homogenates and extracts of tissues. The CLBA is based on the effect of thyroid hormone binding inhibitor (THBI) on partition of a constant amount of radiolabeled ligand [(125I]T4) between fixed amounts of serum and an anti-T4 antibody. The method is convenient, rapid, sensitive, and reproducible. The coefficient of variation averaged 8.9% within an assay and 12.8% between assays. Several fatty acids, e.g. arachidonic acid, lauric acid, linolenic acid, and linoleic acid, had potent THBI activity in the CLBA; arachidonic acid was more potent than the other fatty acids. Since oleic acid cross-reacted substantially with T4-binding sites on anti-T4, its THBI activity was examined by an equilibrium dialysis method; it was about 77% as potent as arachidonic acid. Arachidic, myristic, palmitic, and stearic acids, choleserol, various phospholipids and triglycerides (triolein and tripalmitin) had little or no THBI activity in the CLBA. THBI activity was detected in the sera of 50% (60% when serum T4 was low and 42% when it was normal) of 34 patients with nonthyroid illnesses (NTI) when studied by CLBA and in 59% (67% when serum T4 was low and 53% when it was normal) of patients when determined by the inhibitory ratio (normalized dialysis ratio/normalized binding ratio). THBI values obtained by the CLBA correlated significantly (r = 0.58; P less than 0.001) with those obtained by the inhibitory ratio method. The dose-response curve of an ether extract of pooled sera of hospitalized patients was parallel to that of arachidonic acid in the CLBA. Among various rat tissues, the small intestine had the most THBI activity in both homogenates and ether extracts of homogenates. Ether (2 vol) extracted about 63% of the THBI activity in small intestine homogenate at pH 5.2. THBI activity was demonstrable in all particulate fractions (especially mitochondria and endoplasmic reticulum) of small intestine homogenate; cytosol contained little or no THBI activity. THBI activity changed little after treatment of small intestine homogenate with trypsin or protease inhibitors.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1975

Competitive protein binding radioassay of plasma and urinary unconjugated cortisol.

In order to establish a more reliable profile of the function of the adrenal cortex, a study has been made of this simultaneous estimation of plasma and urinary unconjugated cortisol by competitive protein binding (CPB) radioassay. Both plasma and urinary cortisol were measured by CPB radioassay after solvent extraction. Normal ranges were found for plasma and urinary cortisol. The samples obtained in the morning (8:00 am) established a normal plasma cortisol range of 9.0-21.6 mug% with a mean of 15.3. range for urinary unconjugated cortisol was 12.0-41.5 mug/day (mean 26.9). Dexamethasone suppression and adrenocorticotrophic hormone stimulation of plasma and urinary cortisol were tested with individuals having normal and abnormal adrenal cortical function. RESULTS INDICATED A GOOD CLINICOPATHOLOGICAL CORRELATION. It was concluded that plasma and urinary unconjugated cortisol are reliable laboratory tests for adrenocortical function and would be the methods of choice rather than urinary 17-ketogenic steroids.

17-Ketosteroids↗
PubMedDate not supplied

Discrepant cortisol values: comparison of radioimmunoassay and competitive protein binding technics.

Radioimmunoassays are expected to yield precise values representing the antigen, i.e., insulin, growth, cortisol, etc. employed in producing the antibody. However, in a recent study of a synthetic ACTH which involved the measurement of cortisol levels (1), we found that above 20gamma%, the radioimmunoassay (RIA) yields cortisol values distinctly above those obtained with the competitive protein binding (CPB) technic. We now suggest a possible explanation for this discrepancy and have calculated a correction factor.

Adrenocorticotropic Hormone↗
PubMed1981

[Studies on the measurement of vitamin D derivatives in human plasma. I. A competitive protein binding assay for 25-hydroxyvitamin D in plasma (author's transl)].

A simple and precise method has been developed for the determination of 25-hydroxyvitamin D in 1 ml of human plasma. The method consists of methanol/chloroform extraction, purification by high pressure liquid chromatography and a competitive protein binding assay using vitamin D deficient rat serum. The ethanol extract from vitamin D deficient chick serum was added to the sample before CPBA to eliminate the non-specific interference in the CPBA system as a vitamin D free serum extract. The assay was sensitive to 0.72 ng/ml of plasma. Satisfactory results were obtained in the dilution and recovery tests. The coefficients of variation were 5.8 approximately 9.1% for the within-assay, and 7.4 approximately 10.3% for the between-assay. Plasma concentrations of 25-hydroxyvitamin D in 46 samples of normal human plasma were 21 +/- 10.5 ng/ml (mean +/- SD), and the seasonal variation was demonstrated. Plasma levels for 25-hydroxyvitamin D were high in patients receiving vitamin D2 and low in patients suffering from liver cirrhosis.

25-Hydroxyvitamin D 2↗
PubMed2001

Anions modulate the potency of geranylgeranyl-protein transferase I inhibitors.

We have identified and characterized potent and specific inhibitors of geranylgeranyl-protein transferase type I (GGPTase I), as well as dual inhibitors of GGPTase I and farnesyl-protein transferase. Many of these inhibitors require the presence of phosphate anions for maximum activity against GGPTase I in vitro. Inhibitors with a strong anion dependence were competitive with geranylgeranyl pyrophosphate (GGPP), rather than with the peptide substrate, which had served as the original template for inhibitor design. One of the most effective anions was ATP, which at low millimolar concentrations increased the potency of GGPTase I inhibitors up to several hundred-fold. In the case of clinical candidate l-778,123, this increase in potency was shown to result from two major interactions: competitive binding of inhibitor and GGPP, and competitive binding of ATP and GGPP. At 5 mm, ATP caused an increase in the apparent K(d) for the GGPP-GGPTase I interaction from 20 pm to 4 nm, resulting in correspondingly tighter inhibitor binding. A subset of very potent GGPP-competitive inhibitors displayed slow tight binding to GGPTase I with apparent on and off rates on the order of 10(6) m(-)1 s(-)1 and 10(-)3 s(-)1, respectively. Slow binding and the anion requirement suggest that these inhibitors may act as transition state analogs. After accounting for anion requirement, slow binding, and mechanism of competition, the structure-activity relationship determined in vitro correlated well with the inhibition of processing of GGPTase I substrate Rap1a in vivo.

Adenosine Triphosphate↗
PubMed1990

Fos, Jun and CREB basic-domain peptides have intrinsic DNA-binding activity enhanced by a novel stabilizing factor.

Transcription factors with a 'leucine zipper' (LZ) domain bind to DNA cis-elements, often with palindromic structures. DNA-binding by such factors requires the presence of highly basic regions in the proteins found adjacent to the LZ domain. In order to determine if the observed DNA-binding specificity is programmed by the basic region itself, we developed a competitive binding assay to compare relative binding affinities of synthetic peptides to specific promoter elements. In this report we demonstrate that the basic domains of the oncoproteins Fos, Jun and the transcription factor CREB, possess the structural information necessary to compete for promoter-specific binding. To study the relative binding affinity of the basic motifs to specific promoter elements, we used synthetic peptides to compete for intrinsic Fos/Jun and CREB DNA-binding activity present in HeLa cell nuclear extracts. These studies demonstrate that the basic peptides of both Fos and Jun have higher affinity for the TPA responsive element (TRE) and the cAMP responsive element (CRE) relative to the corresponding peptide for CREB. The peptides showed virtually no affinity for either Sp1 or octamer consensus promoter sequences, demonstrating that these basic peptides also retained their promoter selectivity. We further demonstrate that the conserved dipolar arrangement of two basic amino acid clusters is required for selective, competitive binding. A second conserved feature critical for competitive binding relates to the distance separating the dipolar basic clusters. Our competition binding assay has also helped to identify a novel protein factor, termed ABP (auxilliary bridging protein), which interacts to stabilize Fos and Jun basic domain binding-interaction with specific promoter elements. The data suggest a mechanism in which ABP acts to promote more stable protein-DNA complexes.

Amino Acid Sequence↗
PubMed1975

Association of human alpha1-antitrypsin with anhydrotrypsin.

Unlike in the reaction of anhydrotrypsin and soybean trypsin inhibitor, human alpha1-antitrypsin was found not to form a complex with bovine anhydrotrypsin. This was shown using three different methods: electrophoresis, affinity chromatography on an anhydrotrypsin-coupled Sepharose column and equilibrium competitive binding assay. These results indicate the importance of the active site serine of trypsin in formation of a complex with alpha1-antitrypsin.

Animals↗
PubMed1991

Coupling of a mutated form of the human beta 2-adrenergic receptor to Gi and Gs. Requirement for multiple cytoplasmic domains in the coupling process.

We constructed five genes encoding mutant human beta 2-adrenergic receptor sequence (beta 2AR) which contained 12-22 amino acid substitutions with corresponding sequence from the human alpha 2AAR in order to assess the receptor domains involved in Gs versus Gi recognition and coupling. Mutant beta 2AR with substitutions in the N (S1)- and C-terminal (S2) portions of the third intracellular loop, the proximal cytoplasmic tail (S3), and two combinations thereof (S2,3 and S1,2,3), were stably expressed in Chinese hamster fibrobasts (CHW-1102), as were the human beta 2AR and alpha 2AAR at comparable receptor levels. All mutant receptors with S2 substitutions (i.e. S2, S2,3, S1,2,3) were significantly (approximately 85%) uncoupled from Gs. Upon exposure to pertussis toxin, which uncouples receptors from Gi, S1,2,3 exhibited a 526 +/- 99% increase in agonist-stimulated adenylylcyclase activity compared with a 59 +/- 13% increase with the wild type receptor. This enhanced ability of S1,2,3 to interact with Gs following pertussis toxin treatment indicates that, in the absence of toxin exposure, substantial coupling occurs between the mutant receptor and Gi. Mutant beta 2AR bearing only one or two alpha 2AAR-substituted sequences showed no such enhancement. Forskolin-stimulated enzyme activities were increased by pertussis toxin treatment to similar degrees in all clones examined, indicating that the observed effects are confined to the receptor-mediated pathway. In the absence of GTP, competition binding experiments with S1,2,3, beta 2AR and alpha 2AAR revealed that approximately 40-50% of the receptors formed a high affinity binding state for agonist. Pertussis toxin treatment markedly reduced this to approximately 19% with S1,2,3, while having no effect on beta 2AR and completely eliminating high affinity agonist binding to alpha 2AAR. These results suggest that S1,2,3 interacts with Gi as well as Gs, and that receptor:G protein coupling requires the concerted participation of multiple cytoplasmic receptor domains.

Adenosine Diphosphate Ribose↗
PubMed1989

Coupling of hepatic prostaglandin receptors to adenylate cyclase through a pertussis toxin sensitive guanine nucleotide regulatory protein.

E-series prostaglandins (PGs) inhibit glucagon-stimulated cyclic AMP accumulation in hepatocytes as well as glucagon-stimulated glycogenolysis and fatty acid oxidation. The present study was designed to test the hypothesis that this inhibition occurs via interactions with a plasma membrane PGE2 receptor coupled to adenylate cyclase. PGE2 receptors in rat liver plasma membranes were examined using competitive binding studies [( 3H]PGE2 vs. PGE1). Binding data were analyzed to determine the number of apparent binding sites and the PGE dissociation constant (Kd) at each site. Rat liver plasma membranes contained two classes of binding sites with Kd values of 9.9 X 10(-10) and 8 X 10(-9) M. Addition of the GTP-analog guanyl-5'-6'-imidodiphosphate (0.1 mM) altered the PGE2 binding such that a single class of sites with low affinity (Kd = 4 X 10(-9) M) was observed. Similarly, liver plasma membranes isolated from rats pretreated with pertussis toxin contained only a single class of PGE2 binding sites in the absence of guanyl-5'-6'-imidodiphosphate (Kd = 3.4 X 10(-9) M). PGE2 (10(-10) M) inhibited liver membrane adenylate cyclase activity stimulated by forskolin (by 57%) and glucagon (by 24%). This inhibition was not observed in membranes isolated from rats treated with pertussis toxin. Thus, the present studies demonstrate that PGE binding to its hepatic receptors is regulated by a pertussis toxin sensitive guanine nucleotide binding protein coupled to inhibition of adenylate cyclase.

Adenylate Cyclase Toxin↗
PubMed1975

A comparative study of serum total thyroxine estimation on unextracted serum by radioimmunoassay and by competitive protein binding.

A rapid and precise radioimmunoassay (RIA) for serum total thyroxine (T4) on as little as 1-10 mul of unextracted serum is described. Results in hypothyroidism (overt and borderline), in euthyroid subjects in pregnant and oestrogen-medicated subjects, and in hyperthyroidism (overt and borderline) are compared with the results on the same sera by an established competitive protein binding technique (Ames' Tetralute) on unextracted serum from a different laboratory. The correlation between the two methods was excellent (r= 0.94) and no significant difference between overall appeared to measure total T4 Reliaby in sera containing only 1-3 or 2-6 nmol/1. Both methods predicted the clinical outcome in borderline hypothyroidism and borderline hyperthyroidism equally well and both gave normal results in T3-toxicosis. It is concluded that both techniques reliably measure total T4- RIA appears to have advantages of sensitivity and precision (especially in the hypothroid range), of simplicity, and of low cost.

Binding, Competitive↗
PubMed2002

Biochemical characterization of the human RAD51 protein. II. Adenosine nucleotide binding and competition.

RecA mediated homologous recombination requires cooperative ATP binding and hydrolysis to assume and maintain an active, extended DNA-protein (nucleoprotein) filament. Human RAD51 protein (hRAD51) lacks the magnitude of ATP-induced cooperativity and catalytic efficiency displayed by RecA. Here, we examined hRAD51 binding and ATPase inhibition pattern by ADP and ATP/adenosine 5'-O-(thiotriphosphate) (ATPgammaS). hRAD51 fully saturates with ATP/ATPgammaS regardless of DNA cofactor (K(D) approximately 5 microm; 1 ATP/1 hRAD51). The binding of ADP to hRAD51 appeared bimodal. The first mode was identical to ATP/ATPgammaS binding (K(app1) approximately 3 microm; 1 ADP/1 hRAD51), while a second mode occurred at elevated ADP concentrations (K(app2) > or = 125 microm; >1 ADP/1 hRAD51). We could detect ADP --> ATP exchange in the high affinity ADP binding mode (K(app1)) but not the low affinity binding mode (K(app2)). At low ATP concentrations (<0.3 mm), ADP and ATPgammaS competitively inhibit the hRAD51 ATPase (K(m)((app)) > K(m)). However, at high ATP (>0.3 mm), the hRAD51 ATPase was stimulated by concentrations of ATPgammaS that were 20-fold above the K(D). Ammonium sulfate plus spermidine decreased the affinity of hRAD51 for ADP substantially ( approximately 10-fold) and ATP modestly ( approximately 3-fold). Our results suggest that ATP binding is not rate-limiting but that the inability to sustain an active nucleoprotein filament probably restricts the hRAD51 ATPase.

Adenine Nucleotides↗
PubMed1998

Highly cooperative homodimerization is a conserved property of neural POU proteins.

POU-domain proteins have been shown to play important roles in the development of the nervous, endocrine, and immune systems. However, the distinctive DNA recognition properties of the six major POU subclasses have not been well defined. Here, we have used random oligonucleotide selection and competitive binding assays to determine the optimal DNA recognition elements for the POU-III and POU-VI protein classes, represented by Brn-2 and Brn-5, respectively. The optimal Brn-5 consensus binding sequence GCATAA(T/A)TTAT strongly resembles that previously determined for the POU-IV (Brn-3) class, whereas Brn-2 exhibits highest affinity for non-octamer sites of the form ATG(A/C)AT(A/T)0-2ATTNAT and for octamer sites that contain a full associated heptamer sequence. Brn-2, Brn-3.0, and their invertebrate homologues all exhibit highly cooperative homodimerization on the Brn-2 consensus sequence, demonstrating that cooperative dimerization is a general property of these neural POU proteins. However, modified sites to which Brn-2 binds only as a monomer mediate the transcriptional effects of Brn-2 better than the consensus sequence, demonstrating that dimerization on these sites diminishes the transactivation ability of the protein. Together with the findings of our prior studies these data greatly facilitate the identification of functional POU recognition elements in the regulatory regions of neural genes.

Animals↗
PubMed1976

The simultaneous assay of progesterone, 17-hydroxyprogesterone, and deoxycorticosterone in human plasma by competitive protein binding.

A method is described for the simultaneous measurement of prosterone (P), 17-hydroxyprogesterone (17-OHP) and deoxycorticosterone (DOC) in plasma by competitive protein-binding assay. After extraction, these steroids were purified by Sephadex LH-20 columns. The methods described here is sensitive enough to detect the above-mentioned steroids in as little as 5 ml of plasma under physiological conditions. The mean physiological plasma level (ng/100 ml) of P, 17-OHP and DOC are 18.2+/-1.6 (SE), 136.5+/-11.7, and 8.6+/-1.0 in men, 14.7+/-1.7, 61,2+/-3.6 and 6.0+/-1.2 in women in the follicular phase, and 645.0+/-108.1, 155.2+/-16.9, and 7.5+/-1.2, in the luteal phase of the menstrual cycle. By this method and others, the responses of plasma levels of 7 adrenal steroids (P, 17-OH, doc, corticosterone, deoxycortisol, cortisol and aldosterone) to ACTH infusion (25U i.v. over 8 hours) were investigated in healthy men. The increase of DOC was more pronounced than that of the other steroids.

Adult↗
PubMed1984

Characterization of alpha1 adrenergic receptors in human benign prostatic hyperplasia.

Bladder outlet obstruction in men with benign prostatic hyperplasia is decreased following administration of prazosin, a selective alpha1 adrenergic antagonist. Prazosin presumably binds and antagonizes alpha1 adrenergic receptors on the smooth muscle cells of the prostatic adenoma. This study represents the first identification and characterization of alpha1 adrenergic receptors in the prostate using radioligand receptor binding methods. The binding of [3H] prazosin in homogenates obtained from human prostatic adenomas was saturable and a single high affinity prazosin binding site was identified (Kd = 0.29 +/- 0.09 nM). The alpha1 adrenergic receptor concentration in these homogenates ranged between 0.28 to 2.05 fmol./ mg. wet wt. prostate. The equilibrium dissociation constant and density of prazosin binding sites were similar in different regions of an enucleated prostate suggesting homogeneity of receptor density and receptor binding sites within an adenoma. The receptor density was not directly proportional to the weight of the surgically removed adenoma. The pharmacology of the prazosin binding sites was characterized by competitive binding experiments using [3H] prazosin and several unlabelled adrenergic analogs. The IC50's determined from competitive binding experiments using [3H] prazosin and alpha-methylnorepinephrine, rauwolscine and corynanthine were characteristic of alpha1 adrenergic receptor binding.

Binding Sites↗
PubMed1976

Postnatal development of mouse plasma and brain corticosterone levels: new findings contingent upon the use of a competitive protein-binding assay.

A competitive protein-binding (CPB) assay, suitable for measuring corticosterone levels in 20 mul of mouse plasma or 100 mg of brain, is described. The postnatal development of adrenocortical function was determined in C57BL/10 and DBA/1 mice by CPB assay of basal and stressinduced levels of plasma corticosterone and resting levels of brain corticosterone. Marked increases in both basal and stressed levels of plasma corticosterone were found beginning at day 12 after birth: mean basal levels rose from about 1 mug/u99 ml on day 12 to peak values of about 10-15 mug/100 ml on days 18-20, and then declined by day 30 to the 13-day level of 2.6 mug/100 ml. This pattern differs significantly from results obtained with standard fluorometric assays for corticosterone; it was determined that a major part of this discrepancy is due to the lack of specificity of the fluorometric assay. The developmental change in brain corticosterone was similar to the pattern found in plasma. Only the stress-induced levels of plasma corticosterone showed significant genetic variation, and this did not appear until about one week after the end of the relative stress-nonresponsive period. These findings should be useful in evaluating hypotheses concerning the developmental regulation of adrenocortical function and the action of glucocorticoids in regulating the biochemical differentiation of other tissues.

Age Factors↗
PubMed1975

Interaction of rabbit hemoplexin with copro- and uroporphyrins.

Rabbit hemopexin forms equimolar complexes in vitro with the I and III isomers of both coproporphyrin and uroporphyrin. The apparent dissociation constants (Kd) of these complexes are estimated to be 4-10(-7) M for coproporphyrin-hemopexin and 10(-6) M for uroporphyrin-hemopexin by equilibrium dialysis and quenching of protein fluorescence. Results of competitive binding experiments suggest that all four porphyrins bind at the heme-binding site of hemopexin, and that the relative affinity of rabbit hemopexin for these porphyrins is: deuteroheme greater than coproporphyrin I or III greater than uroporphyrin I or III. These findings provide further evidence that hemopexin may function as a transport protein for circulating coproporphyrins as well as for heme.

Animals↗
PubMed1992

The effects of aging on muscarinic receptor/G-protein coupling in the rat hippocampus and striatum.

In the striatum and hippocampus, there is a loss of sensitivity to muscarinic agonists with age which has been traced to events early in the signal transduction pathway. Our laboratory has therefore focussed on investigations at this level. The current experiments investigate the effects of age on G-protein/receptor interactions by using competitive binding assays to measure the ability of GppNHp to decrease the proportion of receptors bound to G-proteins in the absence and the presence of added Mg2+. L-[3H]Quinuclidinyl benzilate was used as a nonselective ligand and [3H]pirenzepine as an M1 selective ligand. We find that: (1) muscarinic receptors and G-proteins in the striatum appear to become loosely coupled with age, with no change in Mg2+ sensitivity. (2) M1-receptor/G-protein complexes in the hippocampus display increased sensitivity to the presence of Mg2+ with age, with those from old but not young tissue requiring added Mg2+ in order to uncouple. This effect, however, may not be M1 specific.

Aging↗