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1.
The physiological function of many cells is dependent on their ability to adhere via receptors to ligand-coated surfaces under fluid flow. We have developed a model experimental system to measure cell adhesion as a function of cell and surface chemistry and fluid flow. Using a parallel-plate flow chamber, we measured the binding of rat basophilic leukemia cells preincubated with anti-dinitrophenol IgE antibody to polyacrylamide gels covalently derivatized with 2,4-dinitrophenol. The rat basophilic leukemia cells' binding behavior is binary: cells are either adherent or continue to travel at their hydrodynamic velocity, and the transition between these two states is abrupt. The spatial location of adherent cells shows cells can adhere many cell diameters down the length of the gel, suggesting that adhesion is a probabilistic process. The majority of experiments were performed in the excess ligand limit in which adhesion depends strongly on the number of receptors but weakly on ligand density. Only 5-fold changes in IgE surface density or in shear rate were necessary to change adhesion from complete to indistinguishable from negative control. Adhesion showed a hyperbolic dependence on shear rate. By performing experiments with two IgE-antigen configurations in which the kinetic rates of receptor-ligand binding are different, we demonstrate that the forward rate of reaction of the receptor-ligand pair is more important than its thermodynamic affinity in the regulation of binding under hydrodynamic flow. In fact, adhesion increases with increasing receptor-ligand reaction rate or decreasing shear rate, and scales with a single dimensionless parameter which compares the relative rates of reaction to fluid shear.  相似文献   

2.
Dynamic adhesion of cells to surfaces is a vital step in a variety of biochemical and physiological phenomena. Bacterial adhesion is responsible not only for problems associated with biofouling and biofilm formation in the biochemical industry but also in the initiation of certain infectious diseases. In this study, we report the effect of critical parameters, such as receptor and ligand densities and shear rate, on receptor-mediated dynamic bacterial adhesion. Adhesion of a pathogenic strain of Staphylococcus aureus to immobilized collagen was studied. The receptor density on the cell surface was varied by harvesting cells at different growth times and was quantified using flow cytometry. Dynamic adhesion experiments were conducted over a range of physiologically relevant shear rates (50 to 1500 s(-1)) using a parallel-plate flow chamber. Video microscopy coupled with digital image processing was employed to quantify adhesion. A semiquantitative comparison between experimental results and theoretical data obtained using a previously proposed mathematical model was also performed. The results suggest that dynamic adhesion is dependent on receptor density and shear rate, but independent of ligand density. This report demonstrates the feasibility of using bacteria to study fundamental aspects of receptor-mediated dynamic adhesion.  相似文献   

3.
《Insect Biochemistry》1989,19(8):809-814
The interaction of locust high density lipophorin (HDLp) with pieces of fat body tissue was studied at 33°C using a radiolabelled ligand binding assay. Under the assay conditions, binding of tritium-labelled HDLp ([3H]HDLp) was demonstrated to correlate linearly with tissue concentration up to ∼ 7 mg of fat body protein per ml of incubation medium. The [3H]HDLp binding that was displaceable by a 20-fold excess of unlabelled HDLp (which is an approximation of the specific binding) reached equilibrium after ∼ 2 h, whereas low levels of non-displaceable binding increased linearly during this time interval. Analysis of the concentration dependent total binding of [3H]HDLp revealed the presence of a specific binding site with an equilibrium dissociation constant of Kd = 3.1 (±0.5) × 10−7 M and a maximal binding capacity of 9.8 (±0.5) ng μg−1 tissue protein. Competition experiments demonstrated that the affinity of unlabelled HDLp for the binding site is similar to the affinity of [3H]HDLp. Unlabelled low density lipophorin (LDLp), however, was shown to have an approx. 20-fold lower affinity for the binding site.  相似文献   

4.
The aggregation of IgE anchored to high-affinity Fc epsilon receptors on rat basophilic leukemia (RBL) cells by multivalent antigens initiates transmembrane signaling and ultimately cellular degranulation. Previous studies have shown that the rate of dissociation of bivalent and multivalent DNP ligands from RBL cells sensitized with anti-DNP IgE decreases with increasing ligand incubation times. One mechanism proposed for this effect is that when IgE molecules are aggregated, a conformational change occurs that results in an increase in the intrinsic affinity of IgE for antigen. This possibility was tested by measuring the equilibrium constant for the binding of monovalent DNP-lysine to anti-DNP IgE under two conditions, where the cell-bound IgE is dispersed and where it has been aggregated into visible patches on the cell surface using anti-IgE and a secondary antibody. No difference in the equilibrium constant in these two cases was observed. We also measured the rate of dissociation of a monovalent ligand from cell surface IgE under these two conditions. Whereas the affinity for monovalent ligand is not altered by IgE aggregation, we observe that the rate of ligand dissociation from IgE in clusters is slower than the rate of ligand dissociation from unaggregated IgE. These results are discussed in terms of recent theoretical developments concerning effects of receptor density on ligand binding to cell surfaces.  相似文献   

5.
Asn and Gln with an amide group at gamma- and delta-positions, respectively, were substituted for distal His-E7 of bovine myoglobin to establish a system where hydrogen bonding interaction between the distal residue and bound-ligand can be altered by changing donor-acceptor distance. Two mutant myoglobins showed nearly identical (1)H-NMR spectral pattern for resolved heme peripheral side-chain and amino acid proton signals and similar two-dimensional NMR connectivities irrespective of cyanide-bound and -unbound states, indicating that the heme electronic structure and the molecular structure of the active site are not affected by a difference in one methylene group at the E7 position. Chemical exchange rate of Asn-E7 N(delta)H proton in met-cyano myoglobin is larger than that of Gln-E7 N(epsilon)H proton by at least two orders of magnitude, suggesting a considerable difference in the strength of hydrogen bond between the E7 side-chain and bound-ligand, due to the differential donor-acceptor distance between the two mutants. Thus a comparative study between the two proteins provides an ideal system to delineate a relationship between the stabilization of bound-ligand by the hydrogen bond and myoglobin's ligand affinity. The Asn-mutant showed a faster dissociation of cyano ion from met-myoglobin than the Gln-mutant by over 30-fold. Similarly, oxygen dissociation is faster in the Asn-mutant than in the Gln-mutant by approximately 100-fold. Association of cyanide anion to the mutant met-myoglobin was accelerated by changing Gln to Asn by a 4-fold. Likewise, oxygen binding was accelerated by approximately 2-fold by the above substitution. The present findings confirm that hydrogen bonding with the distal residue is a dominant factor for determining the ligand dissociation rate, whereas steric hindrance exerted by the distal residue is a primary determinant for the ligand association.  相似文献   

6.
The contribution of metal ion ligand type and charge to catalysis and regulation at the lower affinity metal ion site (n2 site) of Escherichia coli glutamine synthetase (GS) was tested by mutagenesis and kinetic analysis. The 2 glutamate residues at the n2 site, E129 and E357, were changed to E129D, E129H, E357H, E357Q, and E357D, representing conservative and nonconservative alterations. Unadenylylated and fully adenylylated enzyme forms were studied. The Mn(2+)-KD values, UV-cis and fluorescence emission properties were similar for all mutants versus WTGS, except E129H. For kinetic determinations with both Mn2+ and Mg2+, nonconservative mutants (E357H, E129H, E357Q) showed lower biosynthetic activities than conservative mutants (E129D, E357D). Relative to WTGS, all the unadenylylated Mn(2+)-activated enzymes showed reduced kcat/Km values for ATP (> 7-fold) and for glutamate (> 10-fold). Of the unadenylylated Mg(2+)-activated enzymes, only E129D showed kinetic parameters competitive with WTGS, and adenylylated E129D was a 20-fold better catalyst than WTGS. We propose the n2-site metal ion activates ADP for departure in the phosphorylation of glutamate by ATP to generate gamma-glutamyl phosphate. Alteration of the charge density at this metal ion alters the transition-state energy for phosphoryl group transfer and may affect ATP binding and/or ADP release. Thus, the steady-state kinetic data suggest that modifying the charge density increases the transition-state energies for chemical steps. Importantly, the data demonstrate that each ligand position has a specialized spatial environment and the charge of the ligand modulates the catalytic steps occurring at the metal ion. The data are discussed in the context of the known X-ray structures of GS.  相似文献   

7.
High affinity binding of isoquinolines, such as PK 11195, is a conserved feature of peripheral-type benzodiazepine receptors (PBR) across species. However, species differences in PBR ligand binding have been described based on the affinity for N1-alkyl-1,4-benzodiazepines, such as Ro5-4864. Ro5-4864 binds with high affinity to the rat receptor but has low affinity for the bovine PBR. Photolabeling with an isoquinoline ligand, [3H]PK 14105, identifies a 17-kDa protein, the PBR isoquinoline binding protein (PBR/IBP), in both species. To further elucidate the role of the PBR/IBP in determining PBR benzodiazepine and isoquinoline binding characteristics, the bovine PBR/IBP was cloned and expressed. Using a cDNA encoding a rat PBR/IBP to screen a fetal bovine adrenal cDNA library, a bovine cDNA encoding a polypeptide of 169 residues was cloned. The bovine and rat PBR/IBPs had similar hydropathy profiles exhibiting five potential transmembrane domains. Transfecting the cloned bovine PBR/IBP cDNA into COS-7 cells resulted in an 11-fold increase in the density of high affinity [3H]PK 11195 binding sites which had only low affinity for Ro5-4864. Expression of the bovine PBR/IBP yields a receptor which is pharmacologically distinct from both endogenous COS-7 PBR and the rat PBR based on the affinity for several N1-alkyl-1,4-benzodiazepine ligands. These results suggest the PBR/IBP is the minimal functional component required for PBR ligand binding characteristics and the different protein sequences account for the species differences in PBR benzodiazepine ligand binding.  相似文献   

8.
The interaction between integrin lymphocyte function-associated antigen-1 (LFA-1) and its ligand intercellular adhesion molecule-1 (ICAM-1) is critical in immunological and inflammatory reactions but, like other adhesive interactions, is of low affinity. Here, multiple rational design methods were used to engineer ICAM-1 mutants with enhanced affinity for LFA-1. Five amino acid substitutions 1) enhance the hydrophobicity and packing of residues surrounding Glu-34 of ICAM-1, which coordinates to a Mg2+ in the LFA-1 I domain, and 2) alter associations at the edges of the binding interface. The affinity of the most improved ICAM-1 mutant for intermediate- and high-affinity LFA-1 I domains was increased by 19-fold and 22-fold, respectively, relative to wild type. Moreover, potency was similarly enhanced for inhibition of LFA-1-dependent ligand binding and cell adhesion. Thus, rational design can be used to engineer novel adhesion molecules with high monomeric affinity; furthermore, the ICAM-1 mutant holds promise for targeting LFA-1-ICAM-1 interaction for biological studies and therapeutic purposes.  相似文献   

9.
Kinetics of ligand binding to the type 1 Fc epsilon receptor on mast cells   总被引:2,自引:0,他引:2  
Rates of association and dissociation of several specific monovalent ligands to and from the type I Fc epsilon receptor (Fc epsilon RI) were measured on live mucosal type mast cells of the rat line RBL-2H3. The ligands employed were a monoclonal murine IgE and Fab fragments prepared from three different, Fc epsilon RI-specific monoclonal IgG class antibodies. These monoclonals (designated H10, J17, and F4) were shown previously to trigger mediator secretion by RBL-2H3 mast cells upon binding to and dimerization of the Fc epsilon RI. Analysis of the kinetics shows that the minimal mechanism to which all data can be fitted involves two consecutive steps: namely, ligand binding to a low-affinity state of the receptor, followed by a conformational transition into a second, higher affinity state h of the receptor-ligand complex. These results resolve the recently noted discrepancy between the affinity of IgE binding to the Fc epsilon RI as determined by means of binding equilibrium measurements [Ortega et al. (1988) EMBO J. 7, 4101] and the respective parameter derived from the ratio of the rate constant of rat IgE dissociation and the initial rate of rat IgE association [Wank et al. (1983) Biochemistry 22, 954]. The probability of undergoing the conformational transition differs for the four different Fc epsilon RI-ligand complexes: while binding of Fab-H10 and IgE favors the h state, binding of Fab-J17 and Fab-F4 preferentially maintains the low-affinity 1 state (at 25 degrees C). The temperature dependence of the ligand interaction kinetics with the Fc epsilon RI shows that the activation barrier for ligand association is determined by positive enthalpic and entropic contributions. The activation barrier of the 1----h transition, however, has negative enthalpic contributions counteracted by a decrease in activation entropy. The h----1 transition encounters a barrier that is predominantly entropic and similar for all ligands employed, thus suggesting that the Fc epsilon RI undergoes a similar conformational transition upon binding any of the ligands.  相似文献   

10.
The rate of leukocyte recruitment to and detachment from the vasculature contributes to cellular tethering, rolling, firm adherence, and migration across an endothelium layer. The molecular rates depend on the type and number of bound integrin or selectin adhesion molecules, shear force acting on the bound adhesion molecules, and affinity state of integrins. Although little is known of the effect that the number of adhesion molecules has on leukocyte recruitment, it has been shown that firm adhesion for cells in suspension may be mediated by small numbers of bound adhesion molecules. We studied the disaggregation of aggregates composed of B78H1 cells transfected with human vascular cell adhesion molecule-1 (VCAM-1) and human monoblastoid U937 cells expressing Very Late Antigen-4 (VLA-4). Aggregate disaggregation rates were obtained and compared to dissociation rates for soluble rhVCAM-1 ligand and monoblastoid U937 cells. Under conditions without shear stress, it was found that average cellular disaggregation rates were a factor of 1.3 +/- 0.4 times slower than molecular dissociation rates for the 1 mM Mn(2+) and 1 mM Mn(2+) + 1 mM Ca(2+) conditions. A simple mathematical model was used to predict how much smaller the dissociation constant would be if the number of bonds holding an aggregate varied from one bond to N bonds under conditions without shear stress. The average number of adhesion bonds holding the cell aggregates together was found to be 1.5 +/- 0.7. This suggests that a few bonds were needed to form cellular aggregates and that increased aggregation was related to integrin affinity changes and not due to clustering or increased bond numbers.  相似文献   

11.
VLA-4 (alpha4beta1) is a key integrin in lymphocytes, interacting with endothelial vascular cell adhesion molecule 1 (VCAM-1) on blood vessels and stroma. To dissect the contribution of the two cytoskeletal VLA-4 adaptor partners paxillin and talin to VLA-4 adhesiveness, we transiently knocked them down in Jurkat T cells and primary resting human T cells by small interfering RNA silencing. Paxillin was required for VLA-4 adhesiveness to low density VCAM-1 under shear stress conditions and was found to control mechanical stability of bonds mediated by the alpha4 subunit but did not affect the integrin affinity or avidity to VCAM-1 in shear-free conditions. Talin 1 maintained VLA-4 in a high affinity conformation, thereby promoting rapid VLA-4 adhesion strengthening to VCAM-1 under both shear stress and shear-free conditions. Talin 1, but not paxillin, was required for VLA-4 to undergo optimal stimulation by the prototypic chemokine, CXCL12, under shear stress conditions. Interestingly, talin 1 and paxillin played the same distinct roles in VLA-4 adhesions of primary T lymphocytes, although VLA-4 affinity to VCAM-1 was at least 200-fold lower in these cells than in Jurkat cells. Collectively, our results suggest that whereas paxillin is a mechanical regulator of VLA-4 bonds generated in the absence of chemokine signals and low VCAM-1 occupancy, talin 1 is a versatile VLA-4 affinity regulator implicated in both spontaneous and chemokine-triggered rapid adhesions to VCAM-1.  相似文献   

12.
Three-dimensional neurite outgrowth rates within fibrin matrices that contained variable amounts of RGD peptides were shown to depend on adhesion site density and affinity. Bi-domain peptides with a factor XIIIa substrate in one domain and a RGD sequence in the other domain were covalently incorporated into fibrin gels during coagulation through the action of the transglutaminase factor XIIIa, and the RGD-dependent effect on neurite outgrowth was quantified, employing chick dorsal root ganglia cultured two- and three-dimensionally within the modified fibrin. Two separate bi-domain peptides were synthesized, one with a lower binding affinity linear RGD domain and another with a higher binding affinity cyclic RGD domain. Both peptides were cross-linked into fibrin gels at concentrations up to 8.2 mol of peptide/mol of fibrinogen, and their effect on neurite outgrowth was measured. Both two- and three-dimensional neurite outgrowth demonstrated a bi-phasic dependence on RGD concentration for both the linear and cyclic peptide, with intermediate adhesion site densities yielding maximal neurite extension and higher densities inhibiting outgrowth. The adhesion site density that yielded maximal outgrowth depended strongly on adhesion site affinity in both two and three dimensions, with lower densities of the higher affinity ligand being required (0.8-1.7 mol/mol for the linear peptide versus 0.2 mol/mol for the cyclic peptide yielding maximum neurite outgrowth rates in three-dimensional cultures).  相似文献   

13.
Homotypic adhesion o2 neutrophils stimulated with chemoattractant is analogous to capture on vascular endothelium in that both processes depend on L-selectin and beta 2-integrin adhesion receptors. Under hydrodynamic shear, cell adhesion requires that receptors bind sufficient ligand over the duration of intercellular contact to withstand hydrodynamic stresses. Using cone-plate viscometry to apply a uniform linear shear field to suspensions of neutrophils, we conducted a detailed examination of the effect of shear rate and shear stress on the kinetics of cell aggregation. A collisional analysis based on Smoluchowski's flocculation theory was employed to fit the kinetics of aggregation with an adhesion efficiency. Adhesion efficiency increased with shear rate from approximately 20% at 100 s-1 to approximately 80% at 400 s-1. The increase in adhesion efficiency. Adhesion efficiency increased with shear rate from approximately 20% at 100 s-1 to approximately 80% at 400 s-1. The increase in adhesion efficiency with shear was dependent on L-selectin, and peak efficiency was maintained over a relatively narrow range of shear rates (400-800 s-1) and shear stresses (4-7 dyn/cm2). When L-selectin was blocked with antibody, beta 2-integrin (CD11a, b) supported adhesion at low shear rates (< 400 s-1). The binding kinetics of selectin and integrin appear to be optimized to function within discrete ranges of shear rate and stress, providing an intrinsic mechanism for the transition from neutrophil tethering to stable adhesion.  相似文献   

14.
Three divalent cation binding sites in the integrin β I domain have been shown to regulate ligand binding and adhesion. However, the degree of ligand binding and adhesion varies among integrins. The αLβ2 and α4β7 integrins show an increase in ligand binding affinity and adhesion when one of their ADMIDAS (adjacent to MIDAS, or the metal ion-dependent adhesion site) residues is mutated. By contrast, the α2β1, α5β1, and αIIbβ3 integrins show a decrease in binding affinity and adhesion when their ADMIDAS is mutated. Our study here indicated that integrin αVβ3 had lower affinity when the ADMIDAS was mutated. By comparing the primary sequences of these integrin subunits, we propose that one residue associated with the MIDAS (β3 Ala(252)) may account for these differences. In the β1 integrin subunit, the corresponding residue is also Ala, whereas in both β2 and β7 integrin subunits, it is Asp. We mutated the β3 residue Ala(252) to Asp and combined this mutant with mutations of one or two ADMIDAS residues. The mutant A252D showed reduced ligand binding affinity and adhesion. The ligand binding affinity and adhesion were increased when this A252D mutant was paired with mutations of one ADMIDAS residue. But when paired with mutations of two ADMIDAS residues the mutant nearly abolished ligand-binding ability, which was restored by the activating glycosylation mutation. Our study suggests that the variation of this residue contributes to the different ligand binding affinities and adhesion abilities among different integrin families.  相似文献   

15.
Rapid activation of integrins in response to chemokine-induced signaling serves as a basis for leukocyte arrest on inflamed endothelium. Current models of integrin activation include increased affinity for ligand, molecular extension, and others. In this study, using real-time fluorescence resonance energy transfer to assess alpha(4)beta(1) integrin conformational unbending and fluorescent ligand binding to assess affinity, we report at least four receptor states with independent regulation of affinity and unbending. Moreover, kinetic analysis of chemokine-induced integrin conformational unbending and ligand-binding affinity revealed conditions under which the affinity change was transient whereas the unbending was sustained. In a VLA-4/VCAM-1-specific myeloid cell adhesion model system, changes in the affinity of the VLA-4-binding pocket were reflected in rapid cell aggregation and disaggregation. However, the initial rate of cell aggregation increased 9-fold upon activation, of which only 2.5-fold was attributable to the increased affinity of the binding pocket. These data show that independent regulation of affinity and conformational unbending represents a novel and fundamental mechanism for regulation of integrin-dependent adhesion in which the increased affinity appears to account primarily for the increasing lifetime of the alpha(4)beta(1) integrin/VCAM-1 bond, whereas the unbending accounts for the increased capture efficiency.  相似文献   

16.
An ancestor of avian IgY was the evolutionary precursor of mammalian IgG and IgE, and present day chicken IgY performs the function of human IgG despite having the domain structure of human IgE. The kinetics of IgY binding to its receptor on a chicken monocyte cell line, MQ-NCSU, were measured, the first time that the binding of a non-mammalian antibody to a non-mammalian cell has been investigated (k(+1) = 1.14 +/- 0.46 x 10(5) mol(-1)sec(-1), k(-1) = 2.30 +/- 0.14 x 10(-3) s(-1), and K(a) = 4.95 x 10(7) m(-1)). This is a lower affinity than that recorded for mammalian IgE-high affinity receptor interactions (Ka approximately 10(10) m(-1)) but is within the range of mammalian IgG-high affinity receptor interactions (human: Ka approximately 10(8)-10(9) m(-1) mouse: Ka approximately 10(7)-10(8) m(-1). IgE has an extra pair of immunoglobulin domains when compared with IgG. Their presence reduces the dissociation rate of IgE from its receptor 20-fold, thus contributing to the high affinity of IgE. To assess the effect of the equivalent domains on the kinetics of IgY binding, IgY-Fc fragments with and without this domain were cloned and expressed in mammalian cells. In contrast to IgE, their presence in IgY has little effect on the association rate and no effect on dissociation. Whatever the function of this extra domain pair in avian IgY, it has persisted for at least 310 million years and has been co-opted in mammalian IgE to generate a uniquely slow dissociation rate and high affinity.  相似文献   

17.
We examined the relative contributions of LFA-1, Mac-1, and ICAM-3 to homotypic neutrophil adhesion over the time course of formyl peptide stimulation at shear rates ranging from 100 to 800 s-1. Isolated human neutrophils were sheared in a cone-plate viscometer and the kinetics of aggregate formation was measured by flow cytometry. The efficiency of cell adhesion was computed by fitting the aggregate formation rates with a model based on two-body collision theory. Neutrophil homotypic adhesion kinetics varied with shear rate and was most efficient at 800 s-1, where approximately 40% of the collisions resulted in adhesion. A panel of blocking Abs to LFA-1, Mac-1, and ICAM-3 was added to assess the relative contributions of these molecules. We report that 1) LFA-1 binds ICAM-3 as its primary ligand supporting homotypic adhesion, although the possibility of other ligands was also detected. 2) Mac-1 binding to an unidentified ligand supports homotypic adhesion with an efficiency comparable to LFA-1 at low shear rates of approximately 100 s-1. Above 300 s-1, however, Mac-1 and not LFA-1 were the predominant molecules supporting cell adhesion. This is in contrast to neutrophil adhesion to ICAM-1-transfected cells, where LFA-1 binds with a higher avidity than Mac-1 to ICAM-1. 3) Following stimulation, the capacity of LFA-1 to support aggregate formation decreases with time at a rate approximately 3-fold faster than that of Mac-1. The results suggest that the relative contributions of beta2 integrins and ICAM-3 to neutrophil adhesion is regulated by the magnitude of fluid shear and time of stimulus over a range of blood flow conditions typical of the venular microcirculation.  相似文献   

18.
The objective of this study was to determine the effect of receptor-ligand affinity on the strength of endothelial cell adhesion. Linear and cyclic forms of the fibronectin (Fn) cell-binding domain peptide Arg-Gly-Asp (RGD) were covalently immobilized to glass, and Fn was adsorbed onto glass slides. Bovine aortic endothelial cells attached to the surfaces for 15 min. The critical wall shear stress at which 50% of the cells detached increased nonlinearly with ligand density and was greater with immobilized cyclic RGD than with immobilized linear RGD or adsorbed Fn. To directly compare results for the different ligand densities, the receptor-ligand dissociation constant and force per bond were estimated from data for the critical shear stress and contact area. Total internal reflection fluorescence microscopy was used to measure the contact area as a function of separation distance. Contact area increased with increasing ligand density. Contact areas were similar for the immobilized peptides but were greater on surfaces with adsorbed Fn. The dissociation constant was determined by nonlinear regression of the net force on the cells to models that assumed that bonds were either uniformly stressed or that only bonds on the periphery of the contact region were stressed (peeling model). Both models provided equally good fits for cells attached to immobilized peptides whereas the peeling model produced a better fit of data for cells attached to adsorbed Fn. Cyclic RGD and linear RGD both bind to the integrin alpha v beta 3, but immobilized cyclic RGD exhibited a greater affinity than did linear RGD. Receptor affinities of Fn adsorbed to glycophase glass and Fn adsorbed to glass were similar. The number of bonds was calculated assuming binding equilibrium. The peeling model produced good linear fits between bond force and number of bonds. Results of this study indicate that 1) bovine aortic endothelial cells are more adherent on immobilized cyclic RGD peptide than linear RGD or adsorbed Fn, 2) increased adhesion is due to a greater affinity between cyclic RGD and its receptor, and 3) the affinity of RGD peptides and adsorbed Fn for their receptors is increased after immobilization.  相似文献   

19.
The group of leukocyte integrins CD11a-c/CD18 coordinate disparate adhesion reactions in the immune system through a regulated process of ligand recognition. The participation of the receptor divalent ion binding site(s) in this mechanism of ligand binding has been investigated. As compared with other divalent cations, Mn2+ ions have the unique property to dramatically stimulate the adhesive functions of the leukocyte integrin CD11b/CD18 (Mac-1), expressed on myelo-monocytic cells. This is reflected in a three- to fivefold increased early monocyte adhesion (less than 20 min) to resting, unperturbed endothelial cells, and increased association of CD11b/CD18 with its soluble ligands fibrinogen and factor X. CD11b/CD18 ligand recognition in the presence of Mn2+ ions is specific, time and concentration dependent, and inhibited by anti-CD11b mAb. At variance with Ca(2+)-containing reactions where CD11b/CD18 functions as an inducible receptor activated by adenine nucleotides or chemoattractants, Mn2+ ions induce per se a constitutive maximal ligand binding capacity of CD11b/CD18, that is not further modulated by cell stimulation. Rather than quantitative changes in surface density, Mn2+ ions increase the affinity of CD11b/CD18 for its complementary ligands up to 10-fold, as judged by Scatchard plot analysis of receptor:ligand interaction under these conditions. Furthermore, monocyte exposure to Mn2+ ions induces the expression of activation-dependent neo-antigenic epitopes on CD11b/CD18, selectively recognized by mAb 7E3. These data suggest that in addition to cell-activating stimuli, favorable engagement of divalent ion binding site(s) can provide an alternative pathway to rapidly regulate the receptor affinity of leukocyte integrins.  相似文献   

20.
Binding of histone H1 to DNA is described by an allosteric model   总被引:1,自引:0,他引:1  
Equilibrium binding data were analyzed to characterize the interaction of the linker histone H1 degrees with unmodified T4 phage DNA. Data were cast into the Scatchard-type plot described by McGhee and von Hippel and fit to their eponymous model for nonspecific binding of ligand to DNA. The data were not fit by the simple McGhee-von Hippel model, nor fit satisfactorily by the inclusion of a cooperativity parameter. Instead, the interaction appeared to be well described by Crothers' allosteric model, in which the higher affinity of the protein for one conformational form of the DNA drives an allosteric transition of the DNA to the conformational form with higher affinity (form 2). At 214 mM Na(+), the observed affinity K for an isolated site on unmodified T4 bacteriophage DNA in the form 2 conformation is 4.5 x 10(7) M(-1). The binding constant for an isolated site on DNA in the conformation with lower affinity, form 1, appears to be about 10-fold lower. Binding affinity is dependent on ion concentration: the magnitude of K is about 10-fold higher at 14 mM (5.9 x 10(8) M(-1) for form 2 DNA) than at 214 mM Na(+) concentration.  相似文献   

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