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PubMed1990

High-performance liquid chromatographic postcolumn reaction detection based on a competitive binding system.

Postcolumn reactions are typically employed to improve detection in high-performance liquid chromatography (HPLC) separation techniques. This study proposes the use of competitive binding principles in designing novel postcolumn reaction schemes. The feasibility of this approach was tested by using the HPLC determination of biotin and biocytin as a model system. The effluent from the HPLC column was merged with a reagent stream containing avidin, whose binding sites were occupied by the dye HABA (2-[4'-hydroxyphenylazo]benzoic acid). HABA was displaced by the analytes from the avidin-HABA complex and the free dye was determined with a UV-vis detector at 345 nm. The procedure was optimized with respect to reactor design, reagent concentrations, and the flow rate of reagent solution. Analytical characteristics of the developed procedure were determined and compared with the direct detection of biotin and biocytin at 220 nm. The postcolumn reaction scheme improved the selectivity and sensitivity of the detection of biotin and biocytin while maintaining similar detection limits.

Azo Compounds↗
PubMed1987

Shallow agonist competition binding curves for beta-adrenergic receptors: the role of tight agonist binding.

The beta 2-adrenergic receptors of bovine trapezius muscle membranes demonstrate tight agonist binding as a result of the formation of complexes between agonists, receptors, and Ns. Preincubation of the membranes with (-)-isoproterenol (followed by washing) causes a time- and concentration-dependent decrease in the number of radioligand-binding sites to a plateau value of 41.5 +/- 4%. The affinity of the remaining sites for the radioligand (-)-[3H]dihydroalprenolol is unchanged. This decrease is stable under radioligand binding conditions but is readily reversed in the presence of GTP. The isoproterenol/(-)-[3H]dihydroalprenolol competition binding curves are shallow. Such a phenomenon is usually interpreted in terms of two interconvertible affinity states of the receptor: the high affinity state reflecting the coupling of the agonist X receptor complex to Ns and the low affinity state not interacting with Ns. However, the competition curves undergo time-dependent shifts to the left. This apparent non-equilibrium can be explained by a model in which tight agonist binding to part of the receptor population is included. The usual computerized interpretation of the competition binding curves do not allow the correct evaluation of agonist binding parameters in the presence of tight agonist binding.

Adrenergic beta-Agonists↗
PubMed1984

A competitive binding assay for measurement of heparan sulfate in tissue digests.

Glycosaminoglycans complex with constituents of normal human serum, a finding that was exploited to develop a competitive binding assay for these substances. Heparan sulfate was isolated from renal cortex and radiolabeled with tritiated borohydride. The elution pattern of the radiolabeled material on Sephadex G-25, Bio-Gel P-30, and AG- 1X8 resin was identical to that of unlabeled heparan sulfate. The tritiated heparan sulfate formed radiolabeled precipitates when incubated with serum and zinc acetate. Binding was dose dependent and saturable. Heparin, heparan sulfate, and the chondroitin sulfates, but not hyaluronate or keratan sulfate, competed with the radiolabeled heparan sulfate for binding in a dose-dependent manner. The assay is specific for heparin polysaccharides in chondroitinase ABC-treated samples and is sensitive to microgram quantities.

Binding, Competitive↗
PubMed1996

Measurement of P-glycoprotein expression in multidrug-resistant human neuroblastoma cell lines using self-competitive binding assay.

To date, all reported measurements of multidrug resistance have been semiquantitative. The purpose of the present study is to establish and validate the self-competitive binding assay technique utilizing monoclonal antibody for quantitative estimation of multidrug resistance in tumor cells. This technique is used for P-glycoprotein concentration measurement in BE(2)-C human neuroblastoma cell line and its sublines with primary resistance to colchicine and actinomycin D. Monoclonal antibody MRK-16 was used in this study. It was labeled with iodine-125 (125I) to trace the concentration of antibody-antigen complexes. The binding data were obtained by varying the concentration of the unlabeled antibody. The results were fitted to a model equation to estimate the number of binding sites and antibody-antigen dissociation constant. The P-glycoprotein concentration was significantly higher in the resistant sublines than in the sensitive line. The highest levels were achieved in actinomycin D-resistant cells: 2.1 x 10(6) binding sites/cell versus 5.4 x 10(4) binding sites/cell in the sensitive cells. The consistency of the results was verified by repeating the study three times for each cell line. The binding assay results were confirmed by Western blot experiments performed on the same cell lines.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Rapid colchicine competition-binding scintillation proximity assay using biotin-labeled tubulin.

We have developed a rapid [3H]colchicine competition-binding scintillation proximity assay (SPA) to evaluate antimitotic compounds that bind to the colchicine-binding site on tubulin. The premise of our assay is that compounds will compete with radiolabeled colchicine for the tubulin-binding domain. Biotin-labeled tubulin is incubated first with unlabeled compound and radiolabeled ligand. Streptavidin-labeled SPA beads are added, and the radiolabel associated with tubulin is directly counted with no separation steps. Under our experimental conditions, the dissociation constant of binding (Kd) for colchicine to tubulin was determined to be 1.4 microM, which was consistent with previously reported values. Assay validation was performed by competitively inhibiting [3H]colchicine binding to tubulin with known microtubule inhibitors and comparing their inhibition constants (Ki). Our SPA bead method is a powerful tool since it overcomes the disadvantage of traditional filtration techniques, as there are no separation steps. It is extremely easy to set up, multiple samples can be assayed and supply and labor costs are reduced because of the minimal volume and test reagents used.

Aminophenols↗
PubMed1997

Complex behavior of marine animal tissue extracts in the competitive binding assay of brevetoxins with rat brain synaptosomes.

Brevetoxins are produced by the marine dinoflagellate Ptychodiscus brevis, an organism linked to red tide outbreaks, and the accompanying toxicity to marine animals and to neurotoxic shellfish poisoning in humans. Brevetoxins bind with high affinity to voltage-sensitive sodium channels and cause increased sodium ion conductance and nerve cell depolarization. The brevetoxin competitive binding assay with tritium-labeled brevetoxin 3 (3H-PbTx-3) and rat brain synaptosomes is a sensitive and specific assay for pure brevetoxins. Here we report that extracts of manatee, turtle, fish, and clam tissues contain components that interfere with the assay by cooperative, noncompetitive inhibition of 3H-PbTx-3 specific binding and increased nonspecific binding to synaptosomes. By determining the "apparent" toxin concentration ("[Toxin]") in the extract at several assay concentrations, a reasonable correction for the complex inhibition could be made using a semilog plot to extrapolate [Toxin] to zero extract concentration to obtain [Toxin]0. Spiking 4 extracts with 60 nM PbTx-3 caused [Toxin]0 to increase by 41 +/- 8 nM, indicating that the noncompetitive components did not prevent the assay of toxin but did reduce the accuracy of the result. Fourfold repetition of the assay of 4 samples gave standard deviations of 25 to 60% of the value of [Toxin]0, so the error can be fairly large, especially for samples with little toxin. Purification of an extract with a 1 g sample prep column of C-18 decreased the complex inhibition by about 3-fold but did not eliminate interference in the assay.

Animals↗
PubMed1978

A study of the specificity of Bandeiraea simplicifolia lectin I by competitive-binding assay with blood-group substances and with blood-group A and B active and other oligosaccharides.

The specificity of Bandeiraea simplicifolia lectin I (BS I) has been studied by competitive-binding assays (CBA) using tritium-labeled human B and hog A substances. Blood-group B substances isolated from horse gastric mucosae and from human ovarian-cyst fluids were much better inhibitors of binding of tritiated blood-group B substance to insoluble BS I-Sepharose 2B than were human blood-group A substances from saliva and ovarian-cyst fluid. A and B active blood-group substances showed the same range of potency in inhibiting binding of tritium-labeled hog A substance to BS I-Sepharose 2B. CBA with BS I-Sepharose 2B, labeled human blood-group B substance, and human blood-group A and B active aligosaccharides separated the haptens into two groups differing in slope. Group 1, containing methyl alpha-D-GalNAcp, D-GalNAcp, and an A active pentasaccharide ARL 0.52, with 3, 19, and 25 nmol respectively needed for 50% inhibition of binding, has a lower slope than group 2, which contains alpha-D-GalNAcp-(1 leads to 3)-2-acetamido-2-deoxy-D-galactitol and p-nitrophenyl alpha-D-GalNAcp, with 3 nmol of each required for 50% inhibition of binding, as well as ten glycosides with terminal, nonreducing, alpha-linked D-Galp. The most potent inhibitors of this group were p-nitrophenyl alpha-D-Galp, alpha-D-Galp-(1 leads to 3)-D-Galp, alpha-D-Galp-(1 leads to 6)-D-Glcp, and methyl alpha-D-Galp, with 5, 7.4, 9.6, and 11 nmol respectively needed to inhibit binding by 50%. The difference in slopes was explainable in terms of a recent finding that BS I exists as a mixture of five isolectins composed of two subunits having different specificities; subunit A is most specific for alpha-linked, terminal, nonreducing D-GalNAcp, but it also reacts with alpha-linked, terminal, nonreducing D-Galp, whereas subunit B tends to be more specific for terminal, nonreducing, alpha-linked D-Galp.

ABO Blood-Group System↗
PubMedDate not supplied

Competitive binding assay using fluorescence resonance energy transfer for the identification of calmodulin antagonists.

The ubiquitous calcium regulating protein calmodulin (CaM) has been utilized as a model drug target in the design of a competitive binding fluorescence resonance energy transfer assay for pharmacological screening. The protein was labeled by covalently attaching the thiol-reactive fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC) to an engineered C-terminal cysteine residue. Binding of the environmentally sensitive hydrophobic probe 2,6-anilinonaphthalene sulfonate (2,6-ANS) to CaM could be monitored by an increase in the fluorescence emission intensity of the 2,6-ANS. Evidence of fluorescence resonance energy transfer (FRET) from 2,6-ANS (acting as a donor) to MDCC (the acceptor in this system) was also observed; fluorescence emission representative of MDCC could be seen after samples were excited at a wavelength specific for 2,6-ANS. The FRET signal was monitored as a function of the concentration of calmodulin antagonists in solution. Calibration curves for both a selection of small molecules and a series of peptides based upon known CaM-binding domains were obtained using this system. The assay demonstrated dose-dependent antagonism by analytes known to hinder the biological activity of CaM. These data indicate that the presence of molecules known to bind CaM interfere with the ability of FRET to occur, thus leading to a concentration-dependent decrease of the ratio of acceptor:donor fluorescence emission. This assay can serve as a general model for the development of other protein binding assays intended to screen for molecules with preferred binding activity.

Amino Acid Sequence↗
PubMed2001

TSH-receptor antibody measurement for differentiation of hyperthyroidism into Graves' disease and multinodular toxic goitre: a comparison of two competitive binding assays.

OBJECTIVES: Graves' disease is characterized by stimulating autoantibodies to the TSH-receptor (TRAb). The aim of this study was to compare the performance of a new TRAb assay based on competitive binding to recombinant human TSH-receptors (H-TRAb) with an assay employing purified porcine TSH-receptors (P-TRAb). Furthermore, to evaluate the applicability of the H-TRAb assay to discriminate between patients with hyperthyroidism due to Graves' disease (GD) and multinodular toxic goitre (MNTG). DESIGN AND MEASUREMENTS: H-TRAb and P-TRAb were measured in patients with newly diagnosed hyperthyroidism due to GD (n = 106) and MNTG (n = 94). For comparison, TRAb was measured in patients with primary autoimmune hypothyroidism, euthyroid subjects with an enlarged thyroid gland by ultrasound, and healthy controls (n = 100 for each group). Patients were consecutively included from a population survey. RESULTS: If the cut-off values recommended by the manufacturer for TSH-receptor antibody positivity were used for evaluation, the sensitivity of the H-TRAb assay vs. the P-TRAb assay in diagnosing GD was: 95.3/67.9% (P < 0.001). Specificity was (H/P-TRAb): 99/99%. The sensitivity of P-TRAb was increased if the upper 97.5% limit of measurements in controls was used as cut-off (H-TRAb vs. P-TRAb: 95.3/80.2%, P < 0.001). Specificity (H/P-TRAb): 98/98%. The difference between assay performance may partly be due to a better technical performance of the H-TRAb assay with more reliable results in the low range of measurements. However, even in GD patients with clearly measurable TRAb, 25% had a P-TRAb < 50% of the value expected from the H-TRAb measurement. This suggests that a subgroup of patients produce TRAb with a higher affinity for the human than the porcine TSH receptor. A relatively high proportion of patients with MNTG were TRAb positive (H-TRAb/P-TRAb: 17/9%). Characteristics of H-TRAb positive and negative MNTG patients were compared. There was no difference between size of thyroid gland and number of nodules by ultrasonography. H-TRAb positive patients had significantly higher serum T4 and T3 and a greater number were TPO-Ab positive. CONCLUSIONS: H-TRAb diagnosed Graves' disease with a high sensitivity and specificity than P-TRAb. The high occurrence of TRAb in multinodular toxic goitre might in part reflect an overlap between Graves' disease and multinodular toxic goitre in some patients.

Adult↗
PubMed1999

Competitive binding of calmodulin isoforms to calmodulin-binding proteins: implication for the function of calmodulin isoforms in plants.

In plants, multiple calmodulin (CaM) isoforms exist in an organism which vary in their primary structures in as much as 32 residues out of their 148 amino acids. These CaM isoforms show differences in their expression patterns and/or target enzyme activation ability. To further understand the biological significance of CaM isoforms, we examined whether CaM isoforms act on specific regulatory targets. In gel overlay assays on various soybean tissue extracts, surprisingly, two soybean CaM isoforms (SCaM-1 and SCaM-4) did not show significant differences in their target binding protein profiles, although they exhibited minor differences in their relative target binding affinities. In addition, both SCaM isoforms not only effectively bound five known plant CaMBPs, but also showed competitive binding to these proteins. Finally, immunolocalization experiments with the SCaM proteins in sections of various tissues using specific antibodies revealed similar distribution patterns for the SCaM isoforms except for root tissues, which indicates that the SCaM isoforms are concomitantly expressed in most plant tissues. These results suggest that CaM isoforms may compete for binding to CaMBPs in vivo. This competitive nature of CaM isoforms may allow modulation of Ca(2+)/CaM signaling pathways by virtue of relative abundance and differential target activation potency.

Binding, Competitive↗
PubMed2003

Theoretical studies on competitive binding of caldesmon and myosin S1 to actin: prediction of apparent cooperativity in equilibrium and slow-down in kinetics of S1 binding by caldesmon.

Several laboratories have reported cooperative binding of S1 to actin in the presence of caldesmon. This cooperative binding has been interpreted with a model similar to that proposed for the binding of S1 to regulated actins in which the binding affinity of S1 is controlled by the position of the tropomyosin filaments. In a recent paper [Sen, A., Chen, Y., Yan, B., and Chalovich, J. M. (2001) Biochemistry 40, 5757-64], we showed qualitatively that S1 binding resulted in rapid dissociation of caldesmon from actin or actin-tropomyosin. This suggests that the cooperativity observed in the case of caldesmon is not due to a conformational change in actin-caldesmon but to the displacement of caldesmon. We show in this paper that the pure competitive binding model, in which both S1 and caldesmon are competing for the same binding sites on actin, can simulate quantitatively the effect of caldesmon on both the equilibrium and the kinetics of S1 binding to actin. This model successfully predicts an apparent cooperativity for the binding of S1 to actin-caldesmon without the need to assume multiple actin-caldesmon structures and produces a decreased rate of S1 binding to actin in the presence of caldesmon. This suggests that the inhibitory action of caldesmon on the actin-activated ATPase activity of myosin in solution and on the generation of active force in a contracting muscle may be simply due to the blocking of myosin binding sites on actin by caldesmon.

Actins↗
PubMed2000

Differential binding affinities of PCBs, HO-PCBs, and aroclors with recombinant human, rainbow trout (Onchorhynkiss mykiss), and green anole (Anolis carolinensis) estrogen receptors, using a semi-high throughput competitive binding assay.

A comparative study was undertaken to assess the ability of 44 polychlorinated biphenyls (PCBs), 9 hydroxylated PCBs (HO-PCBs), and 8 aroclors at concentrations ranging from 1 nM to 10 microM to compete with [3H]17beta-estradiol (E2) for binding to bacterially expressed fusion proteins using a semi-high throughput competitive-binding assay. The fusion proteins consisted of the D, E, and F domains of human (alpha), cloned reptilian (Anolis carolinensis) and recloned rainbow trout (Onchorhynkiss mykiss) estrogen receptors (ER) linked to the glutathione S-transferase (GST) protein. GST-hERalphadef (human), GST-aERdef (reptile) and GST-rtERdef (rainbow trout) fusion proteins exhibited high affinity for E2 with dissociation constants (Kd) of 0.4+/-0.1 nM, 0.7+/-0.2 nM, and 0.6+/-0.1 nM, respectively. Of the 44 PCBs examined, only PCBs 104, 184, and 188 effectively competed with [3H]E2 for binding to the GST-rtERdef protein with IC50 values ranging from 0.4-1.3 microM. In contrast, these same congeners only caused a 30% displacement of [3H]E2 in GST-hERalphadef and GST-aERdef proteins. Several additional congeners were found to bind to the GST-rtERdef fusion protein, although the degree of interaction varied among congeners. Among the HO-PCBs, 2',3',4',5'-tetrachloro-4-biphenylol and 2,6,2',6'-tetrachloro-4-biphenylol bound to all three fusion proteins with IC50 values ranging from 0.1-0.3 microM. Dimethyl sulphoxide (DMSO) concentrations of 20% significantly increased the ability of PCBs 104, 184, and 188 to compete with [3H]E2 for binding to the GST-ERdef fusion proteins, whereas at 20% DMSO, a significant reduction in saturable binding was observed. These results demonstrate that ERs from different species exhibit differential ligand preferences and relative binding affinities for PCBs, which can be dramatically affected by DMSO concentration.

Amino Acid Sequence↗
PubMed2000

Comparison of commercial latex agglutination and sandwich enzyme immunoassays with a competitive binding inhibition enzyme immunoassay for detection of antigenemia and antigenuria in a rabbit model of invasive aspergillosis.

A commercial latex agglutination assay (LA) and a sandwich enzyme immunoassay (SEIA) (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France) were compared with a competitive binding inhibition assay (enzyme immunoassay [EIA]) to determine the potential uses and limitations of these antigen detection tests for the sensitive, specific, and rapid diagnosis of invasive aspergillosis (IA). Toward this end, well-characterized serum and urine specimens were obtained by using a rabbit model of IA. Serially collected serum or urine specimens were obtained daily from control rabbits or from rabbits immunosuppressed and infected systemically with Aspergillus fumigatus. By 4 days after infection, EIA, LA, and SEIA detected antigen in the sera of 93, 93, and 100% of A. fumigatus-infected rabbits, respectively, whereas antigen was detected in the urine of 93, 100, and 100% of the rabbits, respectively. False-positive results for non-A. fumigatus-infected rabbits for EIA, LA, and SEIA were as follows: for serum, 14, 11, and 23%, respectively; for urine, 14, 84, and 90%, respectively. Therefore, although the sensitivities of all three tests were similar, the specificity was generally greater for EIA than for LA or SEIA. Infection was also detected earlier by EIA, by which the serum of 53% of A. fumigatus-infected rabbits was positive as early as 1 day after infection, whereas the serum of only 27% of the rabbits tested by LA was positive. Although the serum of 92% of A. fumigatus-infected rabbits was positive by SEIA as early as 1 day after infection, the serum of a high percentage (50%) was false positive before infection. The urine of 21% of A. fumigatus-infected rabbits was positive by EIA as early as 1 day after infection, and the urine of none of the rabbits was false positive before infection. When EIA results for urine specimens were combined with those for serum, sensitivity was improved (i.e., 67% of rabbits were positive by 1 day after infection and only one rabbit gave a false-positive result). A total of 93% of A. fumigatus-infected rabbits were positive for antigen in urine as early as 1 day after infection and the urine of 100% of the rabbits was positive by SEIA. However, before infection, 79% of A. fumigatus-infected rabbits were false positive for antigen in urine by LA and 90% were false positive for antigen in urine by SEIA. These data indicate that the EIA has the potential to be used to diagnose IA with both serum and urine specimens and to detect a greater number of infections earlier with greater specificity than the specificities achieved with the commercial tests.

Animals↗
PubMed2006

Deuterium isotope effects observed during competitive binding chiral recognition electrospray ionization--mass spectrometry of cinchona alkaloid-based systems.

Deuterium isotope effects are reported for binding between tert-butylcarbamoyl-quinine/quinidine chiral selectors and isotopomeric quasienantiomers of N-(3,5-dinitrobenzoyl)leucine measured using electrospray ionization-mass spectrometry (ESI-MS) and competitive binding. Evaluation of mixtures of each selector with one labeled and one unlabeled enantiomeric selectand of identical configuration showed a significant difference in measured ion abundances of diastereomeric complexes between the selector and each selectand. It was found that in some cases, the complex containing the nondeuterated selectand was 15% more abundant than its deuterated counterpart. On the basis of an assessment of solution- and gas-phase isotope effects reported in the literature, a series of control experiments were performed to study the origin of the effects. On the basis of these measurements, our preliminary conclusion is that the differing gas-phase physicochemical nature of the deuterated versus nondeuterated selectand represents the strongest contribution to the observed effect in this chiral molecular recognition system.

Binding, Competitive↗
PubMed1984

Selective degradation of heparin and heparan sulphate, and reactivity of products in the competitive binding assay.

Heparin and heparan sulphate were degraded by various chemical methods, including sodium meta-periodate oxidation, alkaline treatment, Smith degradation, and treatment with acids such as nitrous and hydrochloric acid. The products were assessed by gel filtration and in the competitive binding assay (CBA) developed recently by Dawes and Pepper (3). With the exception of nitrous acid treatment, all the chemical methods were nonselective in their effect on heparin and heparan sulphate. However, nitrous acid treatment exhibited a degree of selectivity which may be useful in enhancing the specificity of CBA. The results also highlighted the significance of sulphated groups in the binding of heparin and heparan sulphate in CBA.

Binding, Competitive↗
PubMed1993

Thrombospondin 1 is a tight-binding competitive inhibitor of neutrophil cathepsin G. Determination of the kinetic mechanism of inhibition and localization of cathepsin G binding to the thrombospondin 1 type 3 repeats.

Thrombospondin 1 was recently shown to bind to and inhibit the activity of neutrophil elastase (Hogg, P. J., Owensby, D. A., Mosher, D. F., Misenheimer, T. M., and Chesterman, C. N. (1993) J. Biol. Chem. 268, 7139-7146). This finding led us to question whether thrombospondin 1 also binds and inhibits the other major serine proteinase of neutrophils, cathepsin G. In a competitive binding assay, cathepsin G bound to thrombospondin 1 reversibly and saturably with a dissociation constant in the low nanomolar range. The kinetic mechanism of inhibition of cathepsin G activity by thrombospondin 1 was determined using the synthetic cathepsin G substrate, Suc-Ala-Ala-Pro-Phe-p-nitroanilide, and is consistent with hyperbolic tight-binding inhibition in which thrombospondin 1 binds cathepsin G and the Michaelis cathepsin G-substrate complex and weakens, but does not abolish, the efficiency of hydrolysis of Suc-Ala-Ala-Pro-Phe-p-nitroanilide. In the presence of 2 mM calcium ions, 2.9 +/- 0.4 mol of cathepsin G interacted with 1 mol of thrombospondin 1 trimer with a site-binding constant of 7.0 +/- 3.5 nM, which reduced the efficiency of hydrolysis of Suc-Ala-Ala-Pro-Phe-p-nitroanilide 8.5 +/- 1.4-fold. A lower limit for the on rate constant of 5 x 10(6) M-1 S-1 was established. The affinity of binding and stoichiometry for the interaction between cathepsin G and thrombospondin 1 was enhanced in the absence of calcium ions. In the presence of EDTA, 5.3 +/- 0.5 mol of cathepsin G interacted with 1 mol of thrombospondin 1 with a site-binding constant of 2.1 +/- 1.6 nM, implying the existence of two binding sites for cathepsin G on each subunit of thrombospondin 1, one or both of which is variably exposed and sensitive to calcium ions. Thrombospondin 1 protected fibronectin from cleavage by cathepsin G and blocked cathepsin G-mediated platelet aggregation. In summary, the binding of cathepsin G to thrombospondin 1 is tight, reversible, and close enough to the active site of cathepsin G to perturb the interactions of a small synthetic substrate and exclude a macromolecular protein substrate and platelets. Using defined proteolytic fragments and different conformers of thrombospondin 1, the binding sites for cathepsin G have been localized to the thrombospondin 1 type 3 repeats.

Amides↗
PubMed1996

Homogeneous bioluminescence competitive binding assay for folate based on a coupled glucose-6-phosphate dehydrogenase--bacterial luciferase enzyme system.

A homogeneous bioluminescence competitive binding assay for folate was developed by using a coupled enzyme system of glucose-6-phosphate dehydrogenase (G6PDH) and bacterial luciferase. A highly substituted G6PDH-folate conjugate was prepared by employing an N-hydroxysuccinimide/carbodiimide method. Folate binding protein inhibits the activity of the conjugate. In the presence of folate, there is a competition between folate and the G6PDH-folate conjugate for the binding site of the folate binding protein, and the activity of the conjugate is recovered. Thus, the concentration of folate can be related to the activity of the G6PDH-folate conjugate, which is directly related to the bioluminescence produced by the coupled enzyme reaction. Using this assay, dose-response curves with a detection limit of 2.5 x 10(-8) M folate were obtained, which is an improvement of an order of magnitude with respect to an assay that monitors G6PDH activity spectrophotometrically. The assay was validated using vitamin tablets and a cell culture medium.

Binding, Competitive↗