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PubMedDate not supplied

Binding kinetics of thrombin and antithrombin III with immobilized heparin using a spacer.

The immobilization of heparin onto polymeric surfaces using a hydrophilic spacer was effective in curtailing surface induced thrombus formation. In this study, the binding kinetics of immobilized heparin with antithrombin III (ATIII) and thrombin were investigated. Low molecular weight heparin (molecular weight, 6,000 daltons) was fractionated on an ATIII affinity column, and it was immobilized onto a styrene/p-amino styrene random co-polymer surface via hydrophilic spacer groups. This polymer substrate was coated onto glass beads (diameter range, 0.088-0.105 mm). PEO (molecular weight 3,400), modified by tolylene diisocyanate, was covalently coupled as a spacer group, followed by heparin. The bioactivity of immobilized heparin was approximately 16.2%, relative to free heparin, and nearly 1:1 binding between heparin and PEO was calculated. The binding constants of immobilized heparin and ATIII, and immobilized heparin and thrombin, were 0.958 x 10(7) M-1 and 1.76 x 10(8) M-1, respectively. The immobilized heparin bound with both ATIII and thrombin, and the binding mechanism was similar to that of free heparin.

Amino Acid Sequence↗

PubMed2004

The influence of glimepiride on the binding kinetics of insulin with its skeletal muscle and liver receptors in rats with short term and prolonged hyperglycemia induced by streptozotocin.

BACKGROUND: The aim of the study was to determine the influence of glimepiride on the binding kinetics of insulin with its skeletal muscle receptor in rats with transient and prolonged hyperglycemia induced by streptozotocin. MATERIAL/METHODS: The studies were performed on healthy male Wistar rats with a body mass of 220+/-30 g, fed with LSM-type standard chow, and given water ad libitum. Transient or prolonged hyperglycemia was induced by intraperitoneal administration of streptozotocin. Blood samples were taken from the right heart ventricle to heparinized test tubes and centrifuged for 10 minutes at 700 i. g. Plasma was collected and the glucose level was determined. From each animal 1 g of skeletal muscle and 1 g of liver were collected as well, placed in liquid nitrogen and stored until determination of the affinity and number of receptors. RESULTS: We found an increase in affinity and binding capacity of high- and low-affinity receptors in rats with both transient and prolonged streptozotocin-induced hyperglycemia. The affinity and binding capacity of receptors increased under the influence of glimepiride in transient hyperglycemia caused by streptozotocin administration. CONCLUSIONS: The affinity and binding capacity of receptors increased under the influence of glimepiride in the course of transient hyperglycemia. The lack of changes in the specific insulin binding and binding capacity of receptors of both low and high affinity in the group of animals with prolonged hyperglycemia requires explanation.

Animals↗

PubMed1993

Plasma protein binding kinetics of valproic acid over a broad dosage range: therapeutic implications.

The aim of the study was to characterize, from the relationship between total and free serum levels of valproic acid obtained over a broad dosage range (10-50 mg/kg), the parameters defining the in-vivo kinetic behaviour of the binding of valproic acid to plasma proteins, their pharmacokinetic and clinical repercussions, and their application to therapeutic drug monitoring (TDM). The study was performed in nine healthy adults (20-35 years) who were given doses of 1000 (group A), 2000 (group B) and 3000 mg (group C) of sodium valproate according to a compensated cross-over design, simultaneously determining the total and free serum levels of valproic acid over a 24-h period. The mean free fraction increases with dose, although this increase is only significant (P < 0.05) for the highest dose (3000 mg). The variation in the free fraction of valproic acid begins to become significant (P < 0.05) at a total drug concentration above 100 mg/l. The mean values of the dissociation constant (K) and binding sites (n) were 460 mumol/l and 1.79, respectively, showing a variability of 86.6 and 38.7%, respectively, and a residual variability of 13.0%. Significant differences (P < 0.05) were found for the total plasma clearance (Cl) but not for the intrinsic plasma clearance (Clu) values, despite their tendency to decrease with the dose. If TDM is to be used for valproic acid, it is the free serum levels that should be determined, especially if high doses are administered, because the total serum levels are not a true reflection of the free ones, as is the case of other anti-epileptic drugs.

Adult↗

PubMed1994

Binding kinetics of fluticasone propionate to the human glucocorticoid receptor.

Receptor-ligand interactions of fluticasone propionate (FP), a glucocorticoid used for inhalation therapy, were determined and compared with dexamethasone, budesonide, and beclomethasone-17-monopropionate, the active metabolite of beclomethasone dipropionate. Two approaches, evaluation of binding kinetics and competition assays, were applied to obtain relative receptor affinities (RRAs) with dexamethasone as reference. A higher association rate constant and a distinctly lower dissociation rate constant for FP compared with the other glucocorticoids resulted in an equilibrium dissociation constant (Kd) of 0.49 nmol/l. Kd dexamethasone was 9.36 nmol/l; derived RRA of FP was 1910. The calculated half-time of the FP-receptor complex was 10 h, thus exceeding the half-times of all other glucocorticoids as well as their RRAs. Competition assays clearly confirmed the rank order of the tested glucocorticoids, although RRAs were generally lower than those found in kinetic assays and strongly dependent on the assay conditions. The high receptor affinity of FP is reflected by clinical trials demonstrating its superiority to other glucocorticoids. For therapeutic application, the long half-time of the FP-receptor complex should support the practicality of longer dose-intervals.

Administration, Topical↗

PubMed2004

Receptor binding kinetics and cellular responses of six N-formyl peptide agonists in human neutrophils.

The goal of this study was to elucidate the relationships between early ligand binding/receptor processing events and cellular responses for the N-formyl peptide receptor system on human neutrophils as a model of a GPCR system in a physiologically relevant context. Binding kinetics of N-formyl-methionyl-leucyl-phenylalanyl-phenylalanyl-lysine-fluorescein and N-formyl-valyl-leucyl-phenylalanyl-lysine-fluorescein to the N-formyl peptide receptor on human neutrophils were characterized and combined with previously published binding data for four other ligands. Binding was best fit by an interconverting two-receptor state model that included a low affinity receptor state that converted to a high affinity state. Response behaviors elicited at 37 degrees C by the six different agonists for the N-formyl peptide receptor were measured. Dose response curves for oxidant production, actin polymerization, and G-protein activation were obtained for each ligand; whereas all ligands showed equal efficacy for all three responses, the ED(50) values varied as much as 7000-fold. The level of agonism and rank order of potencies of ligands for actin and oxidant responses were the same as for the G-protein activation assay, suggesting that the differences in abilities of ligands to mediate responses were determined upstream of G-protein activation at the level of ligand-receptor interactions. The rate constants governing ligand binding and receptor affinity conversion were ligand-dependent. Analysis of the forward and reverse rate constants governing binding to the proposed signaling receptor state showed that it was of a similar energy for all six ligands, suggesting the hypothesis that ligand efficacy is dictated by the energy state of this ligand-receptor complex. However, the interconverting two-receptor state model was not sufficient to predict response potency, suggesting the presence of receptor states not discriminated by the binding data.

Actins↗

PubMed1994

Luminescent oxygen channeling immunoassay: measurement of particle binding kinetics by chemiluminescence.

A method for monitoring formation of latex particle pairs by chemiluminescence is described. Molecular oxygen is excited by a photosensitizer and an antenna dye that are dissolved in one of the particles. 1 delta gO2 diffuses to the second particle and initiates a high quantum yield chemiluminescent reaction of an olefin that is dissolved in it. The efficiency of 1 delta gO2 transfer between particles is approximately 3.5%. The technique permits real-time measurement of particle binding kinetics. Second-order rate constants increase with the number of receptor binding sites on the particles and approach diffusion control. By using antibody-coated particles, a homogeneous immunoassay capable of detecting approximately 4 amol of thyroid-stimulating hormone in 12 min was demonstrated. Single molecules of analyte produce particle heterodimers that are detected even when no larger aggregates are formed.

Antigen-Antibody Reactions↗

PubMed1993

Effects of DSP-4 on monoamine and monoamine metabolite levels and on beta adrenoceptor binding kinetics in rat brain at different times after administration.

Effects of DSP-4 on noradrenaline (NA), 3-methoxy-4-hydroxyphenyl glycol (MHPG), serotonin (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) levels and on beta adrenoceptor binding kinetics (Bmax and KD) in rat hippocampus, cortex and hypothalamus were studied between 24 hours and 14 days after systemic administration. Beta adrenoceptor numbers in hippocampus and cortex, but not in hypothalamus, were significantly increased after DSP-4. No significant changes in KD values were observed in hypothalamus, but significant increases in this parameter were measured in hippocampus and cortex. NA and MHPG levels were significantly decreased in all three brain regions, but MHPG/NA ratios were increased in hippocampus, decreased in cortex and unchanged in hypothalamus. Very prominent increases in 5-HIAA levels were observed in all three brain regions, but only at one day after DSP-4. The greatest increases in 5-HIAA levels occurred in the hippocampus, but this effect of DSP-4 appeared to be slightly diminished by pre-treatment with fluoxetine. In cortex and hippocampus 5-HT levels were slightly, but significantly decreased after DSP-4.

Animals↗

PubMed1994

Interaction of transforming growth factor alpha with the epidermal growth factor receptor: binding kinetics and differential mobility within the bound TGF-alpha.

The interaction of transforming growth factor alpha (TGF-alpha) with the complete extracellular domain of the epidermal growth factor receptor (EGFR-ED) was examined by nuclear magnetic resonance (NMR) spectroscopy. The 1H NMR resonances of the methyl groups of TGF-alpha were used as probes of the interaction of TGF-alpha with the EGF receptor to determine the binding kinetics and the differential mobility within the bound TGF-alpha. The methyl resonances were studied because there are 14 methyl containing residues well dispersed throughout the structure of TGF-alpha and the relaxation properties of methyl groups are well understood. Changes in the longitudinal and transverse 1H NMR relaxation rates of the methyl resonances of TGF-alpha caused by binding to the 85-kDa EGFR-ED were studied. From these measurements it was determined that the interaction was in the NMR fast exchange limit. A binding mechanism to rationalize the different rates determined by NMR and surface plasmon resonance techniques [Zhou, M., et al. (1993) Biochemistry 32, 8193-8198] is proposed. The transverse relaxation rate (R2) enhancements of the various methyl resonances displayed a regional dependence within the bound TGF-alpha molecule. Resonances from the C-terminus of TGF-alpha, which were flexible in the unbound molecule, revealed dramatic increases in their R2 upon binding to the EGFR-ED along with resonances from the interior of TGF-alpha. However, upon binding, the R2 enhancements of the methyl resonances from the N-terminus of TGF-alpha, which were also flexible in the unbound TGF-alpha, were slight; indicating a retention of mobility of this region for bound TGF-alpha. The implications of these data with respect to the mechanism of receptor activation and the design of antagonists are discussed.

Amino Acid Sequence↗

PubMed1995

Ligand binding kinetics of IL-2 and IL-15 to heteromers formed by extracellular domains of the three IL-2 receptor subunits.

Studies on the binding of IL-2 to its receptor (IL-2R) have generally been limited to receptors expressed on cell surfaces. This has hampered detailed kinetic and mechanistic studies at the molecular level. We have prepared the soluble extracellular domains of all three receptor subunits (called alpha, beta and gamma) by recombinant techniques and have used these to perform detailed kinetic studies of their binding properties using the technique of surface plasmon resonance. We describe a novel approach whereby the receptors are assembled on an antibody surface, being held by an epitope engineered into the C-terminus of each of these domains. Thus the receptors are oriented naturally leading to homogeneous ligand binding kinetics. We have characterized the interactions of the heteromeric complexes of these subunits with mouse and human IL-2 and their analogs, as well as the recently discovered cytokine, IL-15. We have also studied the extracellular domains of the mouse receptor subunits for the first time and have used these as well as mouse-human hybrid receptors to probe the mechanism of assembly of these complexes. We show that no additional proteins are required to reproduce the properties of these complexes in vitro. In addition, kinetic studies with site-specific analogs of IL-2 and the mouse-human receptor hybrids clearly indicate that the extracellular domains of alpha and beta can together readily bind ligand with kinetic properties distinct from those of the constituent subunits. In contrast, a complex containing ligand and the extracellular domains of beta and gamma was comparatively difficult to assemble and required prolonged exposure to IL-2. Our method enabled us to calculate the stoichiometry of these complexes and to determine that anchoring these subunits is necessary to efficiently drive complex formation. The kinetic and equilibrium differences between the mouse and human receptor complexes, and between IL-2 and IL-15 binding to these receptors clarify the roles of the alpha and gamma subunits in the differential response of cells to different cytokines that may be present simultaneously in the environment.

Amino Acid Sequence↗

PubMed1992

Differences between aldosterone and its antagonists in binding kinetics and ligand-induced hsp90 release from mineralocorticosteroid receptor.

We have previously reported that mineralocorticosteroid receptor (MR) is a 8-9 S heterooligomeric complex that includes the 90 kDa heat shock protein (hsp90). To elucidate how antagonist-receptor complexes are biologically inactive in terms of transcriptional regulation, we analyzed the binding of mineralocorticosteroid agonists and antagonists with MR and the ligand-induced transformation of its heterooligomeric structure. This study was performed in the cytosol of adrenalectomized rat kidney and of COS cells transiently transfected with human MR cDNA. Although aldosterone antagonists (SC9420 and RU26752) bind MR with the same affinity as aldosterone, they dissociate much more rapidly from the 8-9 S form of both rat and human MR than does aldosterone. Using sedimentation gradient analysis, we showed that the interaction between hsp90 and the steroid binding subunit of MR is highly dependent upon the nature of the steroid ligand since the binding of aldosterone antagonists results in an easy release of hsp90. We propose that both rapid dissociation of ligand and weakened hsp90-receptor interaction play a key role in the mechanism of mineralocorticosteroid antagonism. In the COS cell model, cortisol, described as a weak mineralocorticosteroid agonist, dissociates also more rapidly from human MR than does aldosterone. Our results suggest that ligand binding kinetics and ligand dependent modification in receptor structure are important modulators of MR function as a transcriptional regulatory factor.

Adrenalectomy↗

PubMed2002

Glycosylation of erythropoietin affects receptor binding kinetics: role of electrostatic interactions.

Erythropoietin (EPO) is a cytokine produced by the kidney whose function is to stimulate red blood cell production in the bone marrow. Previously, it was shown that the affinity of EPO for its receptor, EPOR, is inversely related to the sialylation of EPO carbohydrate. To better understand the properties of EPO that modulate its receptor affinity, various glycoforms were analyzed using surface plasmon resonance. The system used has been well characterized and is based on previous reports employing an EPOR-Fc chimera captured on a Protein A surface. Using three variants of EPO containing different levels of sialylation, we determined that sialic acid decreased the association rate constant (k(on)) about 3-fold. Furthermore, glycosylated EPO had a 20-fold slower k(on) than nonglycosylated EPO, indicating that the core carbohydrate also negatively impacted k(on). The effect of electrostatic forces on EPO binding was studied by measuring binding kinetics in varying NaCl concentrations. Increasing NaCl concentration resulted in a slower k(on) while having little impact on k(off), suggesting that long-range electrostatic interactions are primarily important in determining the rate of association between EPO and EPOR. Furthermore, the glycosylation content (i.e., nonglycosylated vs glycosylated, sialylated vs desialylated) affected the overall sensitivities of k(on) to [NaCl], indicating that sialic acid and the glycan itself each impact the overall effect of these electrostatic forces.

Carbohydrate Conformation↗

PubMed1986

The binding kinetics and interaction of DNA fluorochromes used in the analysis of nuclei and chromosomes by flow cytometry.

The interactions and binding characteristics of DNA dyes used in the flow cytometric analysis of chromatin were studied using human chromosomes and mouse thymocyte nuclei. The kinetics of dye binding and the relationship between fluorescence intensity and dye concentration are presented. Under the conditions used, Hoechst 33258, propidium iodide and chromomycin A3 reach an equilibrium with thymocyte nuclei after approximately 5 min, 20 min and more than 1 h, respectively. The same binding kinetics are observed with Hoechst 33258 and chromomycin when nuclei are stained with a mixture of the two dyes. Sodium citrate, which improves the resolution of flow karyotypes, causes a rapid increase in Hoechst and propidium iodide fluorescence, but a decrease in the fluorescence of chromomycin. The relative peak positions of chromosomes in a flow karyotype are unaffected by sodium citrate addition. The spectral interaction between Hoechst and chromomycin is quantified. There is variation among the human chromosome types in the amount of energy transferred from Hoechst to chromomycin. By measuring the Hoechst and chromomycin fluorescence of each chromosome after Hoechst excitation, it is shown that the amount of energy transferred is correlated to the ratio of the amount of Hoechst to chromomycin bound. Although the energy transfer between the two dyes is considerable, this has little effect on the reproducibility of flow karyotype measurements. The relative peak positions of all human chromosomes in a 64 X 64 channel flow karyotype, except for the 13 and Y chromosomes, vary in the order of 0.5 channel over a 16-fold change in either Hoechst or chromomycin concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Evidence for an alternative pathway for colchicine binding to tubulin, based on the binding kinetics of the constituent rings.

The kinetics of tropolone methyl ether binding to tubulin were measured by following the loss of colchicine binding capacity upon preincubation of tubulin with tropolone methyl ether. At 25 degrees C a bimolecular association rate constant of 2.7 (+/- 0.2) M-1 min-1 was determined, and from the temperature dependence an activation energy of 37 (+/- 8) kJ.mol-1 was calculated. By displacement experiments a dissociation rate constant of 2.9 (+/- 0.6) x 10(-2) min-1 was determined at 25 degrees C. The effect of 3',4',5'-trimethoxyacetophenone (TMA) is 2-fold. TMA reduces the apparent association rate constant of colchicine, indicating that it equilibrates very rapidly and reversibly with the colchicine binding site. From this reduction the binding constant for TMA can be obtained. At 25 degrees C a value of 112 (+/- 13) M-1 is estimated. The binding of TMA is practically thermoneutral. Preincubation of tubulin with TMA over 30 min not only reduces the subsequent binding rate constant of colchicine but also the amplitude. This indicates that TMA also binds slowly in a second mode or site. Stopped-flow kinetic studies reveal that fast TMA binding competes for the initial binding of colchicine. From these results it is concluded that colchicine binds initially with its trimethoxybenzene ring and in a subsequent step with the tropolone ring.

Acetophenones↗

PubMed2005

Monitoring the DNA binding kinetics of a binuclear ruthenium complex by energy transfer: evidence for slow shuffling.

The semirigid binuclear ruthenium complex Delta,Delta-[mu-(11,11'-bidppz)(phen)(4)Ru(2)](4+) has been shown to rearrange slowly from an initial groove-bound nonluminescent state to a final intercalated emissive state by threading one of its bulky Ru(phen)(2) moieties through the DNA base stack. When this complex binds to poly[d(A-T)(2)], a further increase in emission from the complex is observed after completion of the intercalation, assigned to reorganization of the intercalated complex. We here report a study of the threading process in poly[d(A-T)(2)], in which the minor groove binding dye DAPI is used as an energy transfer probe molecule to assess the distribution of ruthenium complex during and also after the actual threading phase. The emission from DAPI is found to change with the same rate as the emission from the ruthenium complex, and furthermore, DAPI does not disturb the binding kinetics of the latter, justifying it as a good probe of both the threading and the reorganization processes. We conclude from the change in the emission from both DAPI and the ruthenium complex with time that DAPI-ruthenium interactions are most pronounced during the process of threading of the complex, suggesting that the complexes are initially threaded slightly anticooperatively and thereafter redistribute along the DNA to reach their thermodynamically most favorable distribution. The final distribution is characterized by a small but significant binding cooperativity, probably as a result of hydrophobic interactions between the complex ions despite their tetravalent positive charges. The mechanism of "shuffling" the complex along the DNA chain is discussed, i.e., whether the ruthenium complex remains threaded (requiring sequential base-pair openings) or if unthreading followed by lateral diffusion within the ionic atmosphere of the DNA and rethreading occurs.

DNA↗

PubMed2006

Enzyme fragment complementation binding assay for p38alpha mitogen-activated protein kinase to study the binding kinetics of enzyme inhibitors.

The majority of protein kinase assays used in drug discovery research are enzyme activity assays. These assays are based on the measurement of phosphorylated protein or peptide substrate, which is the end product of the enzyme reaction. Since most kinase inhibitors are ATP competitive, prediction of the activity of compounds in cellular systems based on potency values in enzyme activity assays is complex, as this should take into account the affinity of the enzyme for ATP and the cellular ATP concentration. The fact that some of the most successful kinase inhibitors, such as STI 571 (imatinib mesylate, Gleevec, Novartis Pharmaceuticals, East Hanover, NJ), act through binding to the inactive isoform of the kinase provides another limitation of enzyme activity assays. Binding assays allow separate measurement of compound affinity to active and inactive kinase and do not require ATP or substrate in the reaction. Recently, a non-radioactive kinase binding assay for p38 mitogen-activated protein kinase has become available from DiscoveRx (Fremont, CA). The assay method, called HitHunter, utilizes enzyme fragment complementation of Escherichia coli beta-galactosidase to generate an assay signal by chemiluminescence. We have reconfigured the commercial assay kit to study the binding kinetics of two known reference inhibitors of the alpha-isoform of p38, the pyridinyl imidazole SB 203580 and the diaryl urea BIRB 796. Our data confirm the slow association kinetics of BIRB 796 as compared to SB 203580, which corresponded with the requirement of a relatively long preincubation time to obtain maximal effect in a cellular assay. Although neither of the two compounds showed preference for either active or inactive p38alpha, our data demonstrate that the HitHunter kinase binding assay can be used to select compounds that specifically target inactive kinase.

Animals↗

PubMed2005

T cell receptor binding kinetics required for T cell activation depend on the density of cognate ligand on the antigen-presenting cell.

CD8(+) T cells recognize peptides of eight to nine amino acid residues long in the context of MHC class I molecules on the surface of antigen-presenting cells (APCs). This recognition event is highly sensitive, as evidenced by the fact that T cells can be activated by cognate peptide/MHC complex (pMHC) at extremely low densities (1-50 molecules). High sensitivity is particularly valuable for detection of antigens at low density, such as those derived from tumor cells and intracellular pathogens, which can down-modulate cognate pMHCs from the surface of APCs to evade recognition by the adaptive immune system. T cell activation is only triggered in response to interactions between the T cell receptor (TCR) and the pMHC ligand that reach a specific half-life threshold. However, interactions with excessively long half-lives result in impaired T cell activation. Thus, efficient T cell activation by pMHC on the surface of APCs requires an optimal dwell time of TCR-pMHC interaction. Here, we show that, although this is a requirement at low cognate pMHC density on the APC surface, at high epitope density there is no impairment of T cell activation by extended TCR-pMHC dwell times. This observation was predicted by mathematical simulations for T cell activation by pMHC at different densities and supported by experiments performed on APCs selected for varied expression of cognate pMHC. According to these results, effective T cell activation depends on a complex interplay between inherent TCR-pMHC binding kinetics and the epitope density on the APC.

Antigen-Presenting Cells↗

PubMed2000

Binding kinetics and ligand specificity for the interactions of the C2B domain of synaptogmin II with inositol polyphosphates and phosphoinositides.

Synaptotagmin II (Syt II) is a key protein in the calcium-dependent exocytosis of synaptic vesicles. It contains two domains homologous to the C2 regulatory region of protein kinase C. The C2A domain acts as a calcium sensor, while the C2B domain has high affinity for inositol polyphosphates (InsP(n)()s) and phosphoinositide polyphosphates (PtdInsP(n)()s). We describe the use of a surface plasmon resonance biosensor in determining the binding kinetics of the C2B domain with InsP(n)() and PtdInsP(n) ligands. Biosensor surfaces were prepared with covalently attached Ins(1,4,5)P(3), Ins(1,3,4,5)P(4), and InsP(6) ligands. The interactions of bacterially expressed His(6)-tagged C2B and (C2A+C2B) domains of Syt II were examined in the presence and absence of competing InsP(n)s and PtdInsP(n)s. Both His(6)-C2B and His(6)-(C2A+C2B) exhibited the highest affinity for the Ins(1,3,4,5)P(4)-modified surface with a K(D) value of 6 nM. The His(6)-(C2A+C2B) had a 10-fold lower association rate constant for the InsP(6)-linked surface (k(a) = 4.6 x 10(3) M(-1) s(-1)) than for the Ins(1,3,4,5)P(4)-modified surface (k(a) = 6.8 x 10(4) M(-1) s(-1)). Two water-soluble phosphoinositides, dioctanoyl-PtdIns(3,4,5)P(3) and dioctanoyl-PtdIns(4,5)P(2), were superior to the soluble InsP(n)s in displacing binding to the Ins(1,3,4,5)P(4)-modified surface. The binding of His(6)-C2B and His(6)-(C2A+C2B) to InsP(n) surfaces did not show significant calcium dependence. These data support a model in which the binding of the C2B domain of Syt II to PtdInsP(n)s is important for the docking and/or fusion of the secretory vesicles to the synaptic plasma membrane.

Binding, Competitive↗

PubMed2000

The lipopolysaccharide barrier: correlation of antibiotic susceptibility with antibiotic permeability and fluorescent probe binding kinetics.

Lipopolysaccharide (LPS), the primary lipid on the surface of Gram-negative bacteria, is thought to act as a permeability barrier, making the outer membrane relatively impermeable to hydrophobic antibiotics, detergents, and host proteins. Mutations in the LPS biosynthetic apparatus increase bacterial susceptibility to such agents. To determine how this increased susceptibility is mediated, we have correlated antibiotic susceptibilities of rough (antibiotic resistant) and deep rough (antibiotic susceptible) bacterial strains with antibiotic permeabilities and fluorescent probe binding kinetics for bilayers composed of LPS purified from the same strains. Bilayer permeabilities of two hydrophobic beta-lactam antibiotics were measured by encapsulating the appropriate beta-lactamases in large unilamellar vesicles. In the presence of MgCl(2), permeabilities of LPS bilayers from rough and deep rough bacteria were similar and significantly lower than those of bacterial phospholipids (BPL). Addition of BPL to the LPS bilayers increased their antibiotic permeability to approximately the level of the BPL bilayers. Binding rates of the fluorescent probe bis-aminonaphthylsulfonic acid (BANS) were 2 orders of magnitude slower for both rough and deep rough LPS bilayers compared to that of bilayers composed of BPL or mixtures of LPS and BPL. On the basis of these results and the observation that deep rough bacteria have higher levels of phospholipid on their surface than do rough bacteria (Kamio, Y., and Nikaido, H. (1976) Biochemistry 15, 2561-2569), we argue that the high susceptibility of deep rough bacteria is due to the presence of phospholipids on their surface. Experiments with phospholipid bilayers showed that the addition of PEG-lipids (containing covalently attached hydrophilic polymers) had little effect on permeability and binding rates, whereas the addition of cholesterol reduced permeability and slowed binding to levels approaching those of LPS. Therefore, we argue that the barrier provided by LPS is primarily due to its tight hydrocarbon chain packing (Snyder et al., (1999) Biochemistry 38, 10758-10767) rather than to its polysaccharide headgroup.

Anti-Bacterial Agents↗