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At least 793 records · Page 44Linked to original sources
PubMed1999

Evaluation of lipid exposure of tryptophan residues in membrane peptides and proteins.

Fluorescence quenching is used to gain information on the exposure of tryptophan residues to lipid in membrane-bound proteins and peptides. A protocol is developed to calculate this exposure, based on a comparison of quenching efficiency and of a fluorescence lifetime (or quantum yield) measured for a protein and for a model tryptophan-containing compound. Various methods of analysis of depth-dependent quenching are compared and three universal measures of quenching profile are derived. One of the measures, related to the area under profile, is used to estimate quenching efficiency. The method is applied to single tryptophan mutants of a membrane-anchoring nonpolar peptide of cytochrome b(5) and of an outer membrane protein A. Analysis of quenching of the cytochrome's nonpolar peptide by a set of four brominated lipids reveals a temperature-controlled reversible conformational change, resulting in increased exposure of tryptophan to lipid and delocalization of its transverse position. Kinetic quenching profiles and fluorescence binding kinetics reported by Kleinschmidt et al. (Biochemistry (1999) 38, 5006-5016) were analyzed to extract information on the relative exposure of tryptophan residues during folding of an outer membrane protein A. Trp-102, which translocates across the bilayer, was found to be noticeably shielded from the lipid environment throughout the folding event compared to Trp-7, which remains on the cis side. The approach described here provides a new tool for studies of low-resolution structure and conformational transitions in membrane proteins and peptides.

Bacterial Outer Membrane Proteins↗
PubMed1990

Characterization of [3H]estradiol-17 beta-(beta-D-glucuronide) binding sites in basolateral and canalicular liver plasma membranes.

The specific binding of [3H]estradiol-17 beta-(beta-D-glucuronide) ([3H]E217G) was examined in isolated basolateral (bLPM) and canalicular (cLPM) liver plasma membranes. Two distinct binding sites were identified in each membrane fraction by competition and saturation experiments. Binding parameters obtained from competition studies were: Kd1 = 26 nM, Bmax1 = 0.26 pmol/mg protein; Kd2 = 2.6 microM, Bmax2 = 27 pmol/mg protein for bLPM; and Kd1 = 81 nM, Bmax1 = 0.61 pmol/mg protein; Kd2 = 6.7 microM, Bmax2 = 79 pmol/mg protein for cLPM. Binding parameters obtained from saturation experiments were not significantly different. There was no Na+ requirement for binding. Kinetic dissociation experiments showed that binding was reversible and revealed two components. The dissociation rate constants did not vary with the method of dilution of radioligand, i.e. by "infinite" volume, or excess unlabeled ligand, thus ruling out the possibility of cooperativity. The ability of a series of compounds to inhibit the binding of [3H]E217G was also examined. In bLPM, taurocholate (TC), estrone sulfate (E1SO4) and bromosulfophthalein (BSP) were able to compete with both binding sites, whereas estriol-17 beta-(beta-D-glucuronide) (E317G), estriol-16 alpha-(beta-D-glucuronide) (E316G), testosterone glucuronide (TG), estradiol-3-(beta-D-glucuronide) (E23G), estriol-3-(beta-D-glucuronide) (E(3)3G), cholate and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) were able to inhibit binding to only the low-affinity site. In cLPM, only the cholestatic steroid D-ring glucuronides (E(3)17G, E(3)16G and TG) and TC were able to compete with both sites, whereas the non-cholestatic steroid A-ring glucuronides (E(2)3G and E(3)3G), BSP and DIDS competed for only the low-affinity site. Based on the observed substrate specificities, the low-affinity sites in bLPM and cLPM are postulated to represent multispecific organic anion carriers. The high-affinity site in cLPM may play a role in mediating steroid D-ring glucuronide-induced cholestasis.

Animals↗
PubMed2004

Ribosome affinity and the prolonged molecular postantibiotic effect of cethromycin (ABT-773) in Haemophilus influenzae.

Cethromycin (ABT-773) is a new ketolide currently in clinical trials, for treatment of community acquired respiratory tract infections. The drug is active in vitro and in vivo against Haemophilus influenzae. In this study, the mechanism of action of cethromycin was investigated in H. influenzae. The drug effect was studied using in vitro transcription-translation and whole cell amino acid incorporation. Both cethromycin and erythromycin inhibit protein synthesis with similar potencies; cethromycin, however, had a prolonged molecular postantibiotic effect (PAE) compared with erythromycin which was consistent with previously reported microbiological data. Ribosome binding assay using ribosomes isolated from H. influenzae NP200 revealed that the ribosome binding affinity of cethromycin was more than 20-fold tighter than that of erythromycin. Studies of binding kinetics showed that the tight binding of cethromycin mainly contributed to the 20-fold slower dissociation from cells. Further studies showed cethromycin had a four-fold faster drug accumulation rate than erythromycin. Therefore, the tight binding of cethromycin with ribosomes likely contributed to the faster drug accumulation, slower dissociation from cells and prolonged molecular PAE of cethromycin for H. influenzae.

Anti-Bacterial Agents↗
PubMed1992

Sequential injection immunoassay utilizing immunomagnetic beads.

A novel sequential injection immunoassay (SIIA) method is described which utilizes immunomagnetic beads to investigate short-time antibody binding. The method is versatile and flexible and may therefore be adapted to many different applications. Initial results for a competitive assay are also presented. The immunomagnetic bead reactor is created within the flowing stream by retaining immunomagnetic beads with an electromagnet to form an open tube reactor. Thus, the spent beads may be discharged after each analysis. This eliminates the problems of instability of reaction surfaces and eliminates the need for additional time traditionally required for regeneration of the solid-reacting phase in order to not only save time and increase sampling frequency but also to provide each individual sampling cycle with a fresh, uniform portion of beads. The spent beads are collected off line and may be regenerated later. Short-time binding kinetic studies demonstrate linear initial binding under 1 min, which then begins to reach saturation in approximately 10 min. Competitive binding assays of monoclonal mouse IgG (MRC OX-19) to polyclonal sheep anti-mouse IgG immobilized to the immunomagnetic beads show reproducible linear displacement in 30-120-s reactions. Fluorescence detection is utilized with a detection limit of 155 ng/mL, and since the reaction time is typically 2 min or shorter, the sampling frequency is 30 samples/h.

Animals↗
PubMed2000

Identification of a major heparin and cell binding site in the LG4 module of the laminin alpha 5 chain.

The G domain of the laminin alpha chains consists of five homologous G modules (LG1-5) and has been implicated in various biological functions. In this study, we identified an active site for cell and heparin binding within the laminin alpha5 G domain using recombinant proteins and synthetic peptides. Recombinant LG4, LG5, and LG4-5 modules were generated using a mammalian expression system. The LG4 and LG4-5 modules were highly active for cell binding, whereas the LG5 module alone showed only weak binding. Heparin inhibited cell binding to the LG4-5 module, whereas no inhibition was observed with EDTA or antibodies against the integrin beta(1) subunit. These results suggest that the LG4-5 module interacts with a cell surface receptor containing heparan sulfate but not with integrins. Solid-phase assays and surface plasmon resonance measurements demonstrated strong binding of the LG4 and LG4-5 modules to heparin with K(D) values in the nanomolar range, whereas a 16-fold lower value was determined for the LG5 module. Treatment with glycosidases demonstrated that N-linked carbohydrates on the LG5 module are complex-type oligosaccharides. The LG4-5 module, devoid of N-linked carbohydrates, exhibited similar binding kinetics toward heparin. Furthermore, cell binding was unaffected by removal of N-linked glycosylation. To localize active sites on the LG4 module, various synthetic peptides were used to compete with binding of the tandem module to heparin and cells. Peptide F4 (AGQWHRVSVRWG) inhibited binding, whereas a scrambled peptide of F4 failed to compete binding. Alanine replacements demonstrated that one arginine residue within F4 was important for cell and heparin binding. Our results suggest a critical role of the LG4 module for heparan sulfate-containing receptor binding within the laminin alpha5 chain.

Amino Acid Sequence↗
PubMed1994

Determinant selection of major histocompatibility complex class I-restricted antigenic peptides is explained by class I-peptide affinity and is strongly influenced by nondominant anchor residues.

The contribution of major histocompatibility complex (MHC) class I-peptide affinity to immunodominance of particular peptide antigens (Ags) in the class I-restricted cytotoxic T lymphocyte (CTL) response is not clearly established. Therefore, we have compared the H-2Kb-restricted binding and presentation of the immunodominant ovalbumin (OVA)257-264 (SIINFEKL) determinant to that of a subdominant OVA determinant OVA55-62 (KVVRFDKL). Immunodominance of OVA257-264 was not attributable to the specific T cell repertoire but correlated instead with more efficient Ag presentation. This enhanced Ag presentation could be accounted for by the higher affinity of Kb/OVA257-264 compared with Kb/OVA55-62 despite the presence of a conserved Kb-binding motif in both peptides. Kinetic binding studies using purified soluble H-2Kb molecules (Kbs) and biosensor techniques indicated that the Kon for association of OVA257-264-C6 and Kbs at 25 degrees C was integral of 10-fold faster (5.9 x 10(3) M-1 s-1 versus 6.5 x 10(2) M-1 s-1), and the Koff approximately twofold slower (9.1 x 10(-6) s-1 versus 1.6 x 10(-5) s-1), than the rate constants for interaction of OVA55-62-C6 and Kbs. The association of these peptides with Kb was significantly influenced by multiple residues at presumed nonanchor sites within the peptide sequence. The contribution of each peptide residue to Kb-binding was dependent upon the sequence context and the summed contributions were not additive. Thus the affinity of MHC class I-peptide binding is a critical factor controlling presentation of peptide Ag and immunodominance in the class I-restricted CTL response.

Amino Acid Sequence↗
PubMed1983

[3H]bicuculline methochloride binding to low-affinity gamma-aminobutyric acid receptor sites.

The binding of [3H]bicuculline methochloride (BMC) to mammalian brain membranes was characterized and compared with that of [3H] gamma-aminobutyric acid ([3H]GABA). The radiolabeled GABA receptor antagonist showed significant displaceable binding in Tris-citrate buffer that was improved by high concentrations of chloride, iodide, or thiocyanate, reaching greater than 50% displacement in the presence of 0.1 M SCN-. An apparent single class of binding sites for [3H]BMC (KD = 30 nM) was observed in 0.1 M SCN- for fresh or frozen rat cortex or several regions of frozen and thawed bovine brain. The Bmax was about 2 pmol bound/mg of crude mitochondrial plus microsomal membranes from unfrozen washed and osmotically shocked rat cortex, similar to that for [3H]GABA. Frozen membranes, however, showed decreased levels of [3H]BMC binding with no decrease or an actual increase in [3H]GABA binding sites. [3H]BMC binding was inhibited by GABA receptor specific ligands, but showed a higher affinity for antagonists and lower affinity for agonists than did [3H]GABA binding. Kinetics experiments with [3H]GABA binding revealed that low- and high-affinity sites showed a similar pharmacological specificity for a series of GABA receptor ligands, but that whereas all agonists had a higher affinity for slowly dissociating high-affinity [3H]GABA sites, bicuculline had a higher affinity for rapidly dissociating low-affinity [3H]GABA sites. This reverse potency between agonists and antagonists during assay of radioactive antagonists or agonists supports the existence of agonist- and antagonist-preferring conformational states or subpopulations of GABA receptors. The differential affinities, as well as opposite effects on agonist and antagonist binding by anions, membrane freezing, and other treatments, suggest that [3H]BMC may relatively selectively label low-affinity GABA receptor agonist sites. This study, using a new commercially available preparation of [3H]bicuculline methochloride, confirms the report of bicuculline methiodide binding by Möhler and Okada (1978), and suggests that this radioactive GABA antagonist will be a valuable probe in analyzing various aspects of GABA receptors.

Animals↗
PubMed1983

Theoretical study of hinge bending in L-arabinose-binding protein. Internal energy and free energy changes.

The L-arabinose-binding protein of Escherichia coli is a periplasmic component of the L-arabinose transport system. Its three-dimensional structure has been determined by x-ray diffraction and shown to have two globular domains and a connecting hinge. These structural features enclose a cleft in which the L-arabinose-binding site is located. The flexibility of the protein hinge that allows hinge-bending motion is investigated here by theoretical analysis of the changes in conformational energy and molecular structure that accompany the opening and closing of the cleft. The hinge of the molecule is found to be quite permissive in that only moderate increases in the internal energy occur upon opening the cleft. Solvation changes of charged groups on the cleft-facing surfaces of the lobes are estimated to make important contributions to the overall energetics of the system. The results indicate that an open conformation for the unliganded protein is stabilized by the exposure and solvation of charged groups in the cleft, and that the cleft is induced to close upon ligand binding. This picture is consistent with experimental data on the structure and the binding kinetics of L-arabinose-binding protein, and provides a physical framework for interpreting such data.

Arabinose↗
PubMed1995

Ligand binding analysis of soluble interleukin-2 receptor complexes by surface plasmon resonance.

Knowledge of the kinetic binding characteristics is often critical to the development of ligand/receptor structure-activity relationships. To better understand the contribution of each of the subunits to ligand binding in the multimeric interleukin-2 receptor system, we have previously prepared stable solution complexes of the alpha- and beta-subunits. In this study, we have employed surface plasmon resonance biosensor methodology (BIAcore) to evaluate both the kinetic and equilibrium binding constants for these complexes. The structural nature of the complexes facilitated immobilization on the sensor surfaces in a manner that minimized interference with ligand interactions. The interleukin-2 receptor complex surfaces displayed excellent binding capacity and stability toward regeneration. In all cases where the binding constants were measurable, the values determined for interleukin-2 were in good agreement with those previously determined by other methods. When interleukin-2 analogs with receptor subunit specific mutations were employed, the binding parameters were consistent with the nature of the mutations. The combination of coiled-coil-mediated solution assembly and surface plasmon resonance analysis of ligand binding provides a powerful approach to the study of multimeric cytokine receptor systems.

Artifacts↗
PubMed1996

Analysis by surface plasmon resonance of the influence of valence on the ligand binding affinity and kinetics of an anti-carbohydrate antibody.

The kinetics of ligand binding by Se155-4, an antibody specific for the Salmonella serogroup B O-polysaccharide, were studied by surface plasmon resonance. Because trace amounts of oligomers in Fab and single-chain antibody variable domain (scFv) preparations resulted in biphasic binding profiles that were difficult to analyze, all kinetic measurements were performed on purified monomeric fragments and, for certain mutant scFv, dimeric forms. Results obtained with monomeric forms indicated that the relatively low affinity of the antibody was due to rapid dissociation (koff approximately 0.25 s-1). Dimeric forms generally showed off-rates that were approximately 20-fold slower and a 5-fold increase in association rate constants to approximately 2 x 10(5) M-1 s-1. Although the association phases for scFv dimers showed good curve fitting to a one component interaction model, the dissociation phases were biphasic, presumably because the availability and accessibility of sites on the antigen always leads to some monovalent attachment. The fast off-rate for dimers was the same as the monomer off-rate. Se155-4 IgG off-rates were very similar to those observed for scFv dimer, whereas the onrate was the same as that obtained with Fab and scFv monomer.

Amino Acid Sequence↗
PubMed1993

Kinetic control of Ca(II) signaling: tuning the ion dissociation rates of EF-hand Ca(II) binding sites.

EF-hand Ca(II) binding sites share a conserved architecture and are prevalent in Ca(II) signaling pathways. The ion binding kinetics of these sites are carefully tuned to provide the physiologically appropriate activation and inactivation time scales. Here we examine kinetic tuning by the side chain at the ninth position of the EF-loop. A model is proposed in which both the size and charge of the side chain contribute to kinetic tuning. To test this model, the ninth loop position of the EF-hand-like site in the Escherichia coli D-galactose binding protein has been engineered and the Tb(III) dissociation kinetics of the resulting sites have been analyzed. Substitutions at this position are observed to generate up to 10(4)-fold changes in Tb(III) dissociation rates, with little effect on Tb(III) affinity. Furthermore, the observed pattern of rate changes confirm the model's predictions; long side chains at the ninth loop position yield slow dissociation kinetics as predicted for a steric block, whereas acidic side chains yield slow dissociation kinetics as expected for an electrostatic barrier.

Calcium↗
PubMed1999

Characterisation of the 5-HT receptor binding profile of eletriptan and kinetics of [3H]eletriptan binding at human 5-HT1B and 5-HT1D receptors.

The affinity of eletriptan ((R)-3-(1-methyl-2-pyrrolidinylmethyl)-5-[2-(phenylsulphonyl )ethyl]-1H-indole) for a range of 5-HT receptors was compared to values obtained for other 5-HT1B/1D receptor agonists known to be effective in the treatment of migraine. Eletriptan, like sumatriptan, zolmitriptan, naratriptan and rizatriptan had highest affinity for the human 5-HT1B, 5-HT1D and putative 5-ht1f receptor. Kinetic studies comparing the binding of [3H]eletriptan and [3H]sumatriptan to the human recombinant 5-HT1B and 5-HT1D receptors expressed in HeLa cells revealed that both radioligands bound with high specificity (>90%) and reached equilibrium within 10-15 min. However, [3H]eletriptan had over 6-fold higher affinity than [3H]sumatriptan at the 5-HT1D receptor (K(D)): 0.92 and 6.58 nM, respectively) and over 3-fold higher affinity than [3H]sumatriptan at the 5-HT1B receptor (K(D): 3.14 and 11.07 nM, respectively). Association and dissociation rates for both radioligands could only be accurately determined at the 5-HT1D receptor and then only at 4 degrees C. At this temperature, [3H]eletriptan had a significantly (P<0.05) faster association rate (K(on) 0.249 min(-1) nM(-1)) than [3H]sumatriptan (K(on) 0.024 min(-1) nM(-1)) and a significantly (P<0.05) slower off-rate (K(off) 0.027 min(-1) compared to 0.037 min(-1) for [3H]sumatriptan). These data indicate that eletriptan is a potent ligand at the human 5-HT1B, 5-HT1D, and 5-ht1f receptors and are consistent with its potent vasoconstrictor activity and use as a drug for the acute treatment of migraine headache.

Animals↗
PubMed2000

Kinetics of the initial steps of rabbit psoas myofibrillar ATPases studied by tryptophan and pyrene fluorescence stopped-flow and rapid flow-quench. Evidence that cross-bridge detachment is slower than ATP binding.

The kinetics of the tryptophan fluorescence enhancement that occurs when myofibrils (rabbit psoas) are mixed with Mg-ATP were studied by stopped-flow in different solvents (water, 40% ethylene glycol, 20% methanol) at 4 degrees C. Under relaxing conditions (low Ca(2+)) in water (mu = 0.16 M, pH 7.4) and at high ATP concentrations, the transient was biphasic, giving a k(fast)(max) of 230 s(-)(1) and a k(slow)(max) of 15 s(-)(1). The kinetics of the two phases were compared with those obtained by chemical sampling using [gamma-(32)P]ATP and quenching in acid (P(i) burst experiments: these give unambiguously the ATP cleavage kinetics), or cold Mg-ATP (cold ATP chase: ATP binding kinetics). k(slow) is due to ATP cleavage, as with S1. Interestingly, k(fast) is slower than the ATP binding kinetics. Instead, this constant appears to report ATP-induced cross-bridge detachment from actin because (1) it was identical to the fluorescence transient obtained on addition of ATP to pyrene-labeled myofibrils; (2) when the initial filament overlap in the myofibrils was decreased, the amplitude of the fast phase decreased; (3) there was no fluorescent enhancement upon the addition of ADP to myofibrils. This is different from the situation with S1 or actoS1 where there was also a fast fluorescent ATP-induced transient but whose kinetics were identical to those of the tight ATP binding. To increase the time resolution and to confirm our results, we also carried out transient kinetics in ethylene glycol and methanol. We interpret our results by a scheme in which a rapid equilibrium between attached (AM.ATP) and detached (M.ATP) states is modulated by the fraction of myosin heads in rigor (AM) during the time of experiment.

Adenosine Triphosphatases↗
PubMed1996

Covalent dimerization of CD28/CTLA-4 and oligomerization of CD80/CD86 regulate T cell costimulatory interactions.

T lymphocyte receptors CD28 and CTLA-4 bind costimulatory molecules CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells and regulate T cell activation. While distinct functional roles have been ascribed to each of these molecules, little is known about how they interact. To better characterize these interactions, we have used surface plasmon resonance to perform equilibrium and kinetic binding analyses of extracellular fragments of CD28/CTLA-4/CD80/CD86. We show that CTLA-4 and CD28 binding are both characterized by rapid kinetic on-rates and rapid dissociation rates. Native disulfide-linked homodimers of CD28 and CTLA-4 bound with two kinetically distinct binding sites, one of high avidity and slow dissociation and one of low avidity and more rapid dissociation. Monomeric CTLA-4 bound only with low affinity and rapid dissociation. Therefore, covalent dimerization of CTLA-4 is required for its high avidity binding. Oligomerization of CD80/CD86 is also required for high avidity CTLA-4 binding since CTLA-4 bound with low avidity to monomeric CD86. This contrasts with the ability of CD80/CD86 on antigen-presenting cells to bind CTLA4Ig with high avidity and predicts their organization as oligomers or clusters that permit multivalent binding. Thus, covalent receptor dimerization and ligand oligomerization are two key features of the CD28/CTLA-4/CD80/CD86 receptor system that control ligand binding and may regulate signal transduction by controlling the duration of receptor occupancy.

Abatacept↗
PubMed1982

Decreased protein binding and thiopental kinetics.

Thiopental kinetics and protein binding were determined in seven surgical patients with chronic renal failure and a thiopental free fraction of 28.0 +/- 6.5% (SD) and in seven age- and weight-matched normal surgical patients with a thiopental free fraction of 15.7 +/- 2.4%. Thiopental clearance, based upon total plasma concentrations, rose from 3.2 +/- 0.6 ml/kg/min in the normal group to 4.5 +/- 1.1 ml/kg/min in the chronic renal failure group. Volume of distribution at steady state, also based on total drug concentrations, rose from 1.9 +/- 0.5 l/kg in the normal group to 3.0 +/- 1.0 l/kg in the chronic renal failure group. These changes in clearance and volume of distribution at steady state are secondary to changes in free drug distribution and elimination. When kinetics were calculated from free drug concentrations, the intrinsic clearance, unbound volume of distribution at steady state, and free fraction in tissues in the normal and renal failure groups did not differ substantially. These data suggest that the kinetic changes based on total drug concentrations are secondary to changes in free fraction in plasma. In chronic renal failure patients, the underlying rate and extent of thiopental distribution and elimination are much the same as in normal patients.

Blood Proteins↗
PubMed1997

Altered methylation substrate kinetics and calcium binding of a calmodulin with a Val136-->Thr substitution.

Calmodulin is trimethylated on Lys115 by a specific calmodulin methyltransferase. Previously, it was shown that the cam2 mutant (Ile136-->Thr) of Paramecium has a decreased level of methylated Lys115 [Lukas, T. J., Friedman, M. W., Kung, C. & Watterson, D. M. (1989) Proc. Natl Acad. Sci. USA 86, 7331-7335]. To investigate how this substitution affects calmodulin structure, function and recognition by the calmodulin methyltransferase, a calmodulin with a Thr136 substitution ([Thr136]calmodulin) was expressed in Escherichia coli in an unmethylated form for in vitro enzyme activator, calcium binding and methylation kinetic analyses. [Thr136]calmodulin was indistinguishable from wild-type calmodulin in saturating (1 mM) calcium in its ability to activate calmodulin-dependent enzymes and in its steady-state kinetic properties with isolated calmodulin methyltransferase. However, [Thr136]calmodulin did show two defects: a complete inability to be methylated in the absence of calcium; and defective calcium binding. As a result, an approximate 10-fold shift in the K0.5 values for calcium dependence of enzyme activation (shifted from 1.1 microM to 9.1 microM of Ca2+ for NAD kinase) and methylation (from 0.71 microM to 7.2 microM of Ca2+ in 0.15 M K+, 2 mM Mg2+) were observed. Non-denaturing electrophoresis and Tyr138 spectroscopic measurements suggest a difference in the conformation of the calcium-depleted structures of normal calmodulin and [Thr136]calmodulin. Overall, the results suggest that the mutation in this conserved position in the COOH-terminal hydrophobic core lowers calcium-binding affinity and alters the calcium-depleted structure leading to decreased methylation at physiological Ca2+ concentrations.

Animals↗
PubMed2000

Kinetic analysis of the interaction between recombinant human Fc(epsilon)RIalpha and serum IgEs from allergic patients.

Binding of IgE to the high-affinity IgE receptor (Fc(epsilon)RI) is the essential event for allergic reaction. Although there are many reports on binding kinetics between myeloma IgE and Fc(epsilon)RI, little is known about the kinetics between heterogeneous polyclonal IgE in the serum and Fc(epsilon)RIalpha. To elucidate the binding characteristics of heterogeneous serum IgE, we measured kinetic parameters of binding between IgE from allergic patients and a recombinant ectodomain of the human Fc(epsilon)RIalpha subunit by real-time interaction analysis based on surface plasmon resonance. Purified IgE monomer from the plasma of allergic patients displayed kinetics for the interaction with Fc(epsilon)RIalpha similar to those of myeloma IgE. In the case of crude IgE samples from allergic patients, one of seven specimens showed significantly higher affinity than highly purified IgE, suggesting that it is possible for IgEs in this specimen to form complexes of higher molecular weight.

Dermatitis, Atopic↗
PubMed1986

Insulin binding to bovine mammary membranes: comparison of microsomes versus smooth membranes.

The study was undertaken to determine if membrane preparations of bovine mammary tissue bound insulin. If binding occurred, it was also the intent to compare binding kinetics between microsomes and smooth membranes. Insulin binding to bovine mammary membranes attained equilibrium, was saturable and was specific for insulin. Additional studies showed binding to be pH sensitive and maximal at 10 mM calcium. Binding affinity of insulin to microsomes and smooth membranes was similar, with the exception that smooth membranes bound 1.8 times more insulin per unit of membrane protein than microsomes. Two different methods were used to generate data for kinetic analysis of the insulin-receptor interaction in microsomes. Competitive binding assays (.6 ng [125I]insulin plus 0 to 100 ng insulin) indicated the presence of two binding sites with dissociation constants (Kd) of .32 and 15.8 nM. Direct titration of microsomes with [125I]insulin (.02 to 10 ng/ml) revealed two binding sites with Kd of .017 and .31 nM. The affinity of the second binding site measured by the competitive binding assay method (Kd of 15.8 nM) is low and therefore may not be of physiological importance for insulin action. Insulin appears to bind to two high-affinity receptor sites in bovine mammary microsomes with Kds of .017 nM and .32 nM. These findings show that bovine mammary tissue contains receptors for insulin. In addition, isolation of smooth membranes from microsomes enriches the number of insulin receptors per unit of membrane protein without altering their binding characteristics.

Animals↗