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At least 523 records · Page 29Linked to original sources
PubMed1984

Conformations of adducts and kinetics of binding to DNA of the optically pure enantiomers of anti-benzo(a)pyrene diol epoxide.

Kinetic flow dichroism studies indicate that the (+) enantiomer of 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene physically bound at intercalative-type sites in double-stranded DNA undergoes covalent binding reactions to form adducts at external binding sites. The conformation of the non-covalent complex derived from the (-) stereoisomer is also intercalative in nature, but the conformations of the covalent adducts are heterogeneous and are characterized by both intercalative-type and external conformations. It is suggested that the distinctly higher biological activity of the (+) enantiomer relative to the activity of the (-) enantiomer may be related to the preponderance of 7,8,9-triol benzo(a)pyrene residues covalently linked to deoxyguanine and located at external binding sites in the DNA adducts.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1996

Binding and kinetic properties of HIV-1 reverse transcriptase markedly differ during initiation and elongation of reverse transcription.

We recently showed that primer tRNA3Lys, human immunodeficiency virus type 1 (HIV-1) RNA and HIV-1 reverse transcriptase (RT) form a specific complex of initiation of reverse transcription that can be functionally distinguished from the elongation complex, which can be obtained by substituting an 18mer oligodeoxyribonucleotide (ODN) for the natural primer (Isel et al., 1996). Here, we compared the binding properties and the single and multiple turnover kinetics of HIV-1 RT in the initiation and elongation complexes. Even though the equilibrium dissociation constants of HIV-1 RT are not very different for the two complexes, RT dissociates approximately 200-fold faster from the initiation complex. Furthermore, nucleotide incorporation by the pre-formed primer-template-RT complexes is reduced by a approximately 50-fold factor during initiation of reverse transcription, compared with elongation. As a consequence, processivity of HIV-1 RT in the initiation complex is close to unity, while it increases by four orders of magnitude during elongation, as expected for a replication enzyme. This processivity change is reminiscent of the transition from initiation to elongation of transcription. Furthermore, our results indicate that the post-transcriptional modifications of tRNA3Lys play a role similar to that of the sigma factor in transcription by the Escherichia coli RNA polymerase: they favour the formation of the specific initiation complex but do not affect the polymerization rate of the bound enzyme.

DNA Primers↗
PubMed1996

Biopolymer-surfactant interaction: 4. Kinetics of binding of cetyltrimethyl ammonium bromide with gelatin, hemoglobin, beta-lactoglobulin and lysozyme.

The binding of CTAB with the proteins, gelatin, hemoglobin, beta-lactoglobulin and lysozyme follow first order kinetics and occurs either in two or three distinct stages. The number of stages depends on the overall configuration of the biopolymers. The denatured protein, gelatin has shown three-stage kinetics under all conditions, whereas the native proteins, hemoglobin, beta-lactoglobulin and lysozyme have exhibited two stage kinetics. Heat treated lysozyme in 8 mol dm-3 urea medium has also shown a two-stage kinetics. On the basis of non interacting binding sites on the proteins and independent sequential binding, the rates of reaction have been observed to increase with temperature and follow the trend k1 >> k2 > k3. The interaction of CTA+ with the proteins is both electrostatic and hydrophobic. Hemoglobin has shown maximum reaction rate whereas, beta-lactoglobulin has shown a minimum. The activation parameters for the kinetic process have exhibited almost non-variant delta G++ and delta H++ < T delta S++. The formation of activation complex in the Eyring model is entropy controlled so also the overall kinetics. An isokinetic entropy-enthalpy compensation phenomenon has been observed for the respective kinetic stages.

Biopolymers↗
PubMed1999

Biosensor measurement of the interaction kinetics between insulin-like growth factors and their binding proteins.

The binding kinetics of human insulin-like growth factor binding protein (IGFBP) 1-6 for recombinant human insulin-like growth factor (IGF) I and II were measured and compared in the present study using surface plasmon resonance biosensor technique. Different concentrations of IGFBPs (5-100 nM) were allowed to interact with the immobilized IGF-I or IGF-II on sensor chip surface. Both des(1-3)IGF-I and insulin are known to bind weakly to the IGFBPs and therefore are used as negative controls for the binding experiments. The resultant sensorgrams were analyzed by using simple 1:1 binding model to derive both the association rate (k(a)) and dissociation rate (k(d)) constants for IGFBP-IGF interactions. The k(a) values of IGFBPs are in the range of 1x10(4) to 9x10(5) M(-1) s(-1) for IGF-I and 7x10(3) to 1.7x10(6) M(-1) s(-1) for IGF-II, respectively. The orders of k(a) for both IGF-I and IGF-II are IGFBP-3>IGFBP-5>IGFBP-6>IGFBP-4>IGFBP-2>++ +IGFBP-1. The k(d) values of IGFBPs are in the range of 1.5x10(-5) to 2x10(-4) s(-1) for IGF-I and 3.6x10(-5) to 3.7x10(-4) s(-1) for IGF-II, respectively. The order of k(d) for IGF-I is IGFBP-6>IGFBP-5>IGFBP-4>IGFBP-3>IGFBP-2>++ +IGFBP-1 and that for IGF-II is IGFBP-5>IGFBP-6>IGFBP-2>IGFBP-4>IGFBP-3>++ +IGFBP-1, respectively. The equilibrium affinity constants (K(A)) were calculated based on the ratio of k(a)/k(d) and were more precise than the published literature values based on competitive radioligand binding assays. The systematic study enables a direct comparison on the IGF-binding properties among the various IGFBPs, and the kinetic data provide additional information to delineate the physiological role of different IGFBPs in vivo.

Biosensing Techniques↗
PubMed1977

20beta-Hydroxysteroid oxidoreductase. Kinetics and binding of corticosteroids and corticosteroid-21-aldehydes.

Corticosteroid-21-aldehydes were reduced only at C-20 by 20 beta-hydroxysteroid dehydrogenase (EC 1.1.1.53) of Streptomyces hydrogenans, and the reduction occurred by transfer of hydrogen from the B-side of NADH. A kinetic investigation of cortisol, cortisone, cortexolone, and the 21-aldehydes of each indicated: (a) the magnitude of the Michaelis constant for any substrate was independent of the second substrate concentration; (b) the 21-aldehydes had larger Michaelis constants (5- to 8-fold) and larger maximum velocities (16- to 40-fold) than the steroids from which they were synthesized; (c) the Michaelis constant for NADH, 29 muM, was independent of the steroid substrate. With cortisol and cortisol-21-aldehyde, product inhibition patterns showed only slope effects with steroid product and NAD+, suggesting a "random" mechanism. Inhibition studies with the "poor" substrate cortisol indicated that cortisol and cortisol-21-aldehyde were reduced at the same site. The inhibition constant (180 muM) agreed with the Michaelis constant of cortisol (140 muM). The steroid product, 20beta-hydroxyprogesterone, gives noncompetitive inhibition patterns with respect to NADH and cortisol-21-aldehyde, indicating a separate binding site exists on the enzyme for this inhibitor. The intrinsic protein fluorescence of 20beta-hydroxysteroid dehydrogenase was quenched by NADH (56%) with a dissociation constant of 16 muM. NAD" quenched the protein fluorescence somewhat less (31%) with a dissociation constant of 104 muM. The fluorescence of 2-p-toluidine-6-naphthalene sulfonate is enhanced in the presence of enzyme, and there is a blue shift in the emission wavelength maximum. The enzyme-enhanced 2-p-toluidine-6-naphthalene sulfonate fluorescence is quenched by NAD+ (32%) with a dissociation constant of 128 muM. Corticosteroids and their corresponding 21-aldehydes completely quench the enhanced 2-p-toluidine-6-naphthalene sulfonate fluorescence and this feature can be used to determine enzyme-steroid dissociation constants. Corticosteroid-21-aldehydes and NAD+ dissociation constants determined in this manner agree with values obtained in kinetic measurements. The dissociation constants determined for cortisol, cortisone, cortexolone, progesterone, and 20beta-hydroxyprogesterone were at least 1 order of magnitude greater than the corresponding kinetic constants, and these findings suggest the presence of a kinetically insignificant binding site.

Adrenal Cortex Hormones↗
PubMed2004

Two major grapefruit juice components differ in intestinal CYP3A4 inhibition kinetic and binding properties.

Bergamottin (BG) and 6',7'-dihydroxybergamottin (DHB) are the most abundant furanocoumarins present in grapefruit juice and have been proposed as major intestinal CYP3A4 inhibitors contributing to grapefruit juice-drug interactions. The relative contribution of BG versus DHB to the interaction potential is unclear, in part due to inconsistencies in the literature regarding inhibitory potency. To resolve these inconsistencies, the inhibitory kinetics of each furanocoumarin toward CYP3A4 catalytic activity were systematically characterized using representative probes from two distinct CYP3A4 substrate subgroups (testosterone and midazolam). With human intestinal microsomes, DHB was a substrate-independent reversible (Ki, approximately 0.8 microM) and mechanism-based (KI, approximately 3 microM; kinact, 0.3-0.4 min(-1)) inhibitor of CYP3A4. In contrast, BG was a substrate-dependent reversible inhibitor, with a Ki (13 microM) using midazolam that was 8-fold greater than that using testosterone, but a substrate-independent mechanism-based inhibitor (KI, approximately 25 microM; kinact, approximately 0.35 min(-1)). Similar trends resulted with cDNA-expressed CYP3A4, only the KI values for BG were approximately 10-fold lower than with microsomes. This seemed to reflect a much greater degree of microsomal protein binding by BG compared with DHB. Differential inhibition kinetics and binding properties between BG and DHB could account in part for the apparent in vitro inconsistencies in the literature. Results also emphasize the importance of appropriate substrate selection when designing inhibition studies involving dietary constituents.

Beverages↗
PubMed1994

Differential expression of epidermal growth factor receptor (EGF-R) gene and regulation of EGF-R bioactivity by progesterone and estrogen in the adult mouse uterus.

The present study examined several aspects of epidermal growth factor receptor (EGF-R) in the mouse uterus during the peri-implantation period and after ovarian hormone treatment of adult ovariectomized mice. The cell-specific distribution, regulation of expression, and binding kinetics were assessed by immunohistochemistry, Northern blot analysis, and ligand binding assays, respectively. Affinity cross-linking studies ascertained the size of the EGF-R, and its bioactivity was examined by determining EGF-dependent subcellular protein tyrosine kinase activity and receptor autophosphorylation. In the intact uterus and separated cell types, EGF-R was detected in the stroma, deciduum, and myometrium, but not in the luminal or glandular epithelium. Uterine EGF-R mRNA transcript profiles showed some differences between pregnant and ovariectomized mice regardless of steroid hormone treatments. Two major [6.5- and 2.7-kilobase (kb)] and two less abundant (9.6- and 5.0-kb) transcripts were detected in pregnant uterine poly(A)+ RNA. Three additional transcripts (< 2.0 kb) were detected in decidual poly(A)+ RNA, and a larger transcript (8.0 kb) was detected in uterine poly(A)+ RNA isolated from ovariectomized mice. Scatchard analysis of EGF binding also revealed apparent differences in binding kinetics between pregnant and ovariectomized mice, although EGF was cross-linked to a 170-kilodalton protein under these conditions. Two classes (Kd, approximately 0.2 and approximately 2.0 nM) of binding sites were noted in pregnant mice, whereas a single class (Kd, approximately 1.0 nM) was found in ovariectomized mice. 17 beta-Estradiol (E2) caused a rapid transient upregulation of uterine EGF-R mRNA levels and increased the number of EGF-binding sites in ovariectomized mice, as did an injection of progesterone (P4). However, the bioactivity of EGF-R could not be detected in uteri of ovariectomized mice treated with oil or P4. E2 treatment was found to be essential for EGF-R bioactivity. Taken together, the results suggest that in the adult mouse uterus, EGF-R status is influenced by factors other than P4 and E2, the epithelium is not the direct target for the actions of EGF-related growth factors as thought previously, the mitogenic effects of these growth factors on epithelial cells in vivo are perhaps mediated by other uterine cell-types expressing EGF-R, and, lastly, these growth factors are not likely to be functional in the uterus in the absence of estrogen. The present observations are supportive of the concept of paracrine or juxtacrine interactions between EGF-related growth factor ligands of luminal epithelial origin and blastocyst EGF-R in the process of implantation.

Animals↗
PubMed1999

Steroidogenic factor 1-DNA binding: a kinetic analysis using surface plasmon resonance.

Basal expression of the glycoprotein hormone alpha-subunit gene in pituitary gonadotrophs is partially dependent on a gonadotroph specific element (GSE) which binds the nuclear receptor, steroidogenic factor-1 (SF-1). We have used surface plasmon resonance (SPR) to determine the association (kappa ass), dissociation (kappa diss) and affinity (KA) constants of SF-1 binding to immobilized oligonucleotides containing either the GSE consensus motif or a GSE mutant with a 2 bp substitution in the GSE site (GSEMUT). In vitro translated SF-1 protein bound the consensus GSE with a threefold increase in affinity constant (P<0.01) compared with the GSEMUT. This was due primarily to a significant increase (P<0.05) in the kappa ass for SF-1 to the GSE and a slower kappa diss (P<0.05). The binding interaction was specific and could be significantly inhibited (P<0. 001) by either anti-SF-1 antibody or excess non-biotinylated GSE. The addition of 14 bp wild-type flanking sequences significantly reduced the affinity of SF-1 to both the GSE (P<0.05) and the GSEMUT (P<0.01). This was due to a significant (P<0.01) decrease in kappa ass for the wild-type and mutant long oligonucleotides compared with the short GSE. Nuclear extracts from alphaT3-1 gonadotroph cells also bound the GSE and GSEMUT, giving kappa diss values which were two- to threefold slower than those obtained with in vitro translated SF-1. Thus, SPR is a powerful technique for examining kinetic interaction between SF-1 and its binding site, and is able to demonstrate the effects of mutations and flanking sequences on that interaction.

Base Sequence↗
PubMed1993

Positron emission tomographic studies of [11C]MDL 72222, a potential 5-HT3 receptor radioligand: distribution, kinetics and binding in the brain of the baboon.

The drug MDL 72222, a selective 5-HT3 receptor antagonist, was labelled with 11C and evaluated for distribution kinetics in brain and in vivo binding to 5-HT3 receptors using cold MDL 72222 challenge and positron emission tomography (PET), in three anaesthetized baboons. After tracer doses of [11C]MDL 72222 (i.v. bolus), 11C radioactivity was equally partitioned between plasma and blood cells and readily crossed the blood-brain barrier; it was distributed heterogeneously into 17 different structures of the brain. The kinetic curves for 11C in tissue showed a rapid initial uptake, followed by a slower ascending phase, up to about the twentieth minute and by a plateau, until the end of experiment (90 min). The plateau values indicated marked uptake in brain which, however, varied according to the region considered. In inhibition studies with cold MDL 72222 (1 mg.kg-1) as pretreatment, co-injection or displacement, no clear-cut effects on the kinetics of [11C] MDL 72222 in brain were detected in any region, including those known to be rich in 5-HT3 receptors. These observations suggest that specific binding to 5-HT3 receptors was not detectable in brain in vivo, because of the high lipophilicity (thus a great capacity for non-specific binding) of MDL 72222. These negative findings may also result from both the possible suboptimal affinity of MDL 72222 for 5-HT3 receptors in vivo and the relatively low density of 5-HT3 receptors present only in selected areas of the mammalian brain. This study is a step in the search of selective 5-HT3 receptor radioligands, adequate for in vivo applications. Slow clearance of [11C]MDL 72222 from brain tissue in baboons, should be accounted for in clinical pharmacokinetic investigations for optimal posology considerations.

Animals↗
PubMed1993

Antigen-antibody binding and mass transport by convection and diffusion to a surface: a two-dimensional computer model of binding and dissociation kinetics.

The kinetics of binding and dissociation between a soluble analyte and an immobilized ligand on or near a surface are described numerically by an iterative computer model. The model is applied to a microflow chamber which is used for surface plasmon resonance measurements. It calculates diffusion perpendicular to the surface, flow parallel to the surface, and the interaction between any number of soluble and immobilized species. If the reaction between analyte and ligand is fast, binding and dissociation are influenced by the transport of the analyte to or away from the surface. In this case the measurement yields apparent association and dissociation rate constants which are not identical with the reaction rate of analyte and ligand. The transition between mass transport-controlled processes and reaction-controlled processes is described and attention is drawn to possible misinterpretations of experimental binding and dissociation curves. The measurement of rate constants higher than allowed by the conventional technique can be performed by elution of the analyte with a second analyte of low molecular weight.

Antibodies↗
PubMed2001

A novel exosite on coagulation factor VIIa and its molecular interactions with a new class of peptide inhibitors.

A new inhibitory peptide binding exosite on the protease domain of coagulation Factor VIIa (FVIIa) has been identified. A novel series of peptide inhibitors of FVIIa, termed the "A-series" peptides, identified from peptide phage libraries and exemplified by peptide A-183 [Dennis, M. S., Roberge, M., Quan, C., and Lazarus, R. A. (2001) Biochemistry 40, 9513-9521], specifically bind at a site that is distinct from both the active site and the exosite of another recently described peptide inhibitor of FVIIa, E-76 [Dennis, M. S., Eigenbrot, C., Skelton, N. J., Ultsch, M. H., Santell, L., Dwyer, M. A., O'Connell, M. P., and Lazarus, R. A. (2000) Nature 404, 465-4701. Peptide A-183 prolonged TF-dependent clotting in human, but not rabbit plasma. Thus, a panel of human FVIIa mutants, containing 70 of the 76 rabbit sequence differences in the protease domain, localized the binding site to residues in the 60s loop and the C-terminus. The location of the exosite was refined by a series of FVIIa alanine mutants, which showed that proximal residues Trp 61 and Leu 251 were critical for binding. Kinetic and equilibrium binding constants for zymogen FVII, FVIIa and TF x FVIIa were determined using immobilized N-terminal biotinylated A-183 by surface plasmon resonance. No peptide binding to nine other human serine proteases was observed. Key residues on the peptide were determined from binding to FVIIa and inhibition of FX activation using a series of alanine mutants of A-183 fused to the Z domain of protein A. Analysis of the mutagenesis data is presented in the context of a crystal structure of A-183 in complex with a version of zymogen FVII [Eigenbrot, C., Kirchhofer, D., Dennis, M. S., Santell, L., Lazarus, R. A., Stamos, J., and Ultsch, M. H. (2001) Structure 9, 627-636]. The shape and proximity of this exosite to the active site may lend itself towards the design of new anticoagulants that inhibit FVIIa.

Amino Acid Sequence↗
PubMed1986

A study of the cerebral cortex cholecystokinin receptor using two radiolabelled probes: evidence for a common CCK 8 and CCK 4 cholecystokinin receptor binding site.

This study was directed at the issue of whether or not subpopulations of cholecystokinin (CCK) receptors exist within the CNS. This was achieved through the use of two radiolabelled probes, namely [125I] Bolton-Hunter (BH) CCK 8 and [3H]pentagastrin (Boc-beta-Ala CCK 4), in comparative studies under identical conditions. Both probes bound with high affinity to the mouse cerebral cortical CCK receptor binding site with apparent equilibrium dissociation constants (KD) of 1.9 nM and 1.4 nM for [3H]pentagastrin and [125I]BH CCK 8, respectively. The maximal binding capacity was 1.05 and 1.15 pmol/g weight for the tritium and iodinated probes, respectively. Hill analysis yielded Hill numbers close to unity, suggesting the absence of more than one binding site and the lack of cooperativity of CCK receptor binding. Kinetic studies revealed binding site homogeneity in that no evidence of multiphasic dissociation curves was seen. Computerised analysis of displacement binding data using LIGAND established that both radiolabelled probes bound to a single site, with the one-site model providing the best fit of the data. Similar rank orders of potency were obtained for various fragments of CCK 8 in competing for the CCK receptor, labelled with either probe. Both CCK 8 and CCK 4 bound with roughly equinanomolar affinity. These studies demonstrate that both CCK 8 and its shorter C-terminal fragment CCK 4 bind to a single class of high-affinity binding site, with as yet no evidence of CNS CCK receptor multiplicity.

Animals↗
PubMed1989

Characterization of insulin binding in the UMR-106 rat osteoblastic osteosarcoma cell.

The correlation of insulin receptor occupancy with classic insulin effects, such as stimulation of glucose uptake, have not been examined in osteoblastlike cells. Accordingly, we characterized insulin binding and examined its relationship to stimulation of glucose analog transport in the UMR-106 rat osteoblastic osteosarcoma cell line. Insulin binding in UMR-106 cells was found to be pH sensitive, temperature dependent, saturable, and specific. Proinsulin was 100-fold less effective than insulin in displacing specific [125I]insulin binding in these cells, whereas IGF-I at concentrations between 0.1 and 10 nM produced no displacement of [125I]insulin but did produce significant displacement of insulin binding at 100 and 1000 nM. Insulin receptor downregulation was observed after exposure to 100 nM insulin for 6 h at 37 degrees C and was temperature dependent. Insulin binding was reversible after 24 h at 4 degrees C. Insulin binding correlated directly with stimulation of 2-deoxyglucose uptake at insulin concentrations between 0.1 and 100 nM, with a half-maximal concentration (ED50) of 0.9 nM for both [125I]insulin binding displacement and stimulation of 2-deoxyglucose uptake. Hence, there was no evidence for spare insulin receptors with regard to stimulation of glucose analog transport. Scatchard analysis of insulin binding kinetics yielded a curvilinear plot, suggesting negative cooperativity. Analysis of insulin binding kinetics using a two-site model yielded a KD of 0.9 nM for the apparent high-affinity binding site and an estimated 80,000 high-affinity binding sites per cell. These findings demonstrate that osteoblastlike cells exhibit a relationship between insulin binding and glucose transport stimulation that is similar to that in liver cells and other insulin-sensitive tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2000

Kinetics of binding properties of 5'-GMP with cisplatin under simulated physiological conditions by capillary electrophoresis.

Kinetics of the adduct formation of cisplatin and its hydrolysis products with 5'-GMP was investigated using capillary electrophoresis (CE) under 'simulated physiological conditions'. Therefore, chloride ion and phosphate concentration as well as the pH were chosen comparable to intracellular conditions. Furthermore, the influence of the sulfur-containing alpha-amino acids L-methionine and L-cysteine on the kinetics of the adduct formation have been studied. Both amino acids increased the half-time of the reaction significantly but did not influence the formation of the major adduct cis-[Pt(NH3)2(N7-GMP)2]2-. The reaction products were separated using CE, and characterised using UV and NMR spectroscopy.

Chlorides↗
PubMed1978

Prediction of the concentration of penicillins in ascitic fluid from serum kinetics and protein binding of the antibiotics in serum and ascitic fluid of dogs.

Ascites was induced in dogs by partial ligation of the inferior vena cava. Concentrations of ampicillin, penicillin G, oxacillin, cloxacillin, dicloxacillin, nafcillin, and methicillin in ascitic fluid and serum were each determined in three animals. All antibiotics were administered intramuscularly in a dose of 15 mg/kg (dry weight) for both single-dose and multiple-dose (eight doses at 4-hr intervals) studies. Binding of antibiotics to serum and ascitic fluid proteins was measured by ultracentrifugation. After single doses the highly protein-bound drugs (oxacillin, cloxacillin, dicloxacillin, and nafcillin) had lower percentages of penetration (ratio of peak in ascitic fluid to that in serum, multipled by 100) than did methicillin, ampicillin, and penicillin G, which have a lower degree of protein binding. The effect was partially overcome by repetitive doses, but five to six doses were usually required to reach equilibrium. In addition to protein binding, serum kinetics (particularly the log mean total concentration of drug in serum after multiple doses) were important determinants of antibiotic concentrations in ascitic fluid. The total antibiotic concentration in ascitic fluid at equilibrium can be accurately calculated from the log mean serum concnetration and the percentages of protein binding in serum and ascitic fluid.

Ampicillin↗
PubMed2005

Development of rationally designed affinity-based drug delivery systems.

Many drug delivery systems have been developed to provide sustained release of proteins in vivo. However, the ability to predict and control the rate of release from delivery systems is still a challenge. Toward this goal, we screened a random drug-binding peptide library (12 amino acids) to identify peptides of varying (i.e. low, moderate, and high) affinity for a model polysaccharide drug (heparin). Peptide domains of varying affinity for heparin identified from the library were synthesized using standard solid phase chemistry. A mathematical model of drug release from a biomaterial scaffold containing drug-binding peptide domains identified from the library was developed. This model describes the binding kinetics of drugs to the peptides, the diffusion of free drug, and the kinetics of enzymatic matrix degradation. The effect of the ratio of binding sites to drug, the effect of varying the binding kinetics and the rate of enzymatic matrix degradation on the rate of drug release was examined. The in vitro release of the model drug from scaffold containing the peptide drug-binding domains was measured. The ability of this system to deliver and modulate the biological activity of protein drugs was also assessed using nerve growth factor (NGF) in a chick dorsal root ganglia (DRG) neurite extension model. These studies demonstrate that our rational approach to drug delivery system design can be used to control drug release from tissue-engineered scaffolds and may be useful for promoting tissue regeneration in vivo.

Affinity Labels↗
PubMed1992

Scintillation proximity assay to study the interaction of epidermal growth factor with its receptor.

Scintillation Proximity Assay (SPA), which does not require the physical separation of receptor bound and free ligand, was applied to study the interaction of Epidermal Growth Factor (EGF) with its receptor (EGFR) in membrane preparations from human placenta. Fluomicrospheres to which the monoclonal anti-EGFR antibody R1 was coupled, were used. Kinetic binding data of the association of 125I-labeled EGF binding to the receptor at 20 degrees C could be fitted according to a double exponential model, which is consistent with the presence of fast and slow associating EGF binding sites. Dissociation kinetics revealed that perturbation of equilibrium conditions rapidly occurs upon washing. Multiple point Scatchard analysis of equilibrium 125I-labeled EGF binding data revealed curvilinearity, indicating the presence of both high and low affinity EGF binding sites. We conclude that SPA is an interesting new tool in the exploration of the interaction of ligands with their receptors, which allows detailed ligand-receptor studies under precise in situ conditions.

Epidermal Growth Factor↗