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B Baird

Publications and source records attributed to B Baird.

At least 19 recordsLinked to original sources
PubMed1992

Dynamic conformations compared for IgE and IgG1 in solution and bound to receptors.

Dynamic conformations of two distinct immunoglobulin (Ig) isotypes, murine IgE and human IgG1, were examined with fluorescence resonance energy transfer measurements. The IgE mutant epsilon/C gamma 3* and the IgG1 mutant gamma/C gamma 3* each bind [5-(dimethylamino)naphthalen-1-yl]sulfonyl (DNS) in two identical antigen binding sites at the amino (N)-terminal ends of the Ig in the Fab segments. Eosin-DNS bound in these Fab sites served as the acceptor probe in these studies. Both Ig have a carboxy (C)-terminal domain (C gamma 3*) which contains genetically introduced cysteine residues. Modification of these cysteine sulfhydryls with fluorescein maleimide provided donor probes near the C-terminal ends of the Ig in the Fc segment. Energy transfer between the C-terminal and N-terminal ends was compared for these two Ig in solution and when they were found to their respective high-affinity receptors on plasma membranes: IgE-Fc epsilon RI on RBL cell membranes and IgG1-Fc gamma RI on U937 cell membranes. Previous energy-transfer measurements with these probes yielded an average end-to-end distance of 71 A for IgE in solution and 69 A for IgE bound to Fc epsilon RI, indicating that in both situations IgE is bent such that the axes of the Fab segments and the axis of the Fc segment do not form a planar Y-shape [Zheng, Shopes, Holowka, & Baird (1991) Biochemistry 30, 9125]. In the current study we found the average end-to-end distance for IgG1 in solution is 75 A and greater than or equal to 85 A for IgG1 bound to Fc gamma RI, suggesting an average bend conformation for IgG1 as well. The contributions of segmental flexibility to the average distances were assessed directly by measuring the efficiency of energy transfer as a function of variations in donor quantum yield caused by a collisional quencher and using these data to extract a Gaussian distribution of end-to-end distances. The distribution average (rho) and half-width (hw) were determined to be as follows: rho = 75 A, hw = 24 A for IgE in solution; rho = 71 A, hw = 12 A for IgE bound to Fc epsilon RI; and rho = 100 A, hw = 88 A for IgG in solution.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Aggregation of IgE-receptor complexes on rat basophilic leukemia cells does not change the intrinsic affinity but can alter the kinetics of the ligand-IgE interaction.

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.

Animals↗
PubMed1992

Rotational motion of monomeric and dimeric immunoglobulin E-receptor complexes.

Erythrosin 5'-thiosemicarbazide labeled immunoglobulin E (IgE) was used to monitor the rotational dynamics of monomeric and dimeric Fc epsilon RI receptors for IgE on rat basophilic leukemia (RBL) basophilic leukemia (RBL) cells using time-resolved phosphorescence anisotropy. Receptors were studied both on living RBL cells and on membrane vesicles derived from RBL cell plasma membrane. The un-cross-linked IgE-receptor complexes on cells and vesicles exhibit rotational correlation times that are consistent with those expected for freely rotating monomers, but a small fraction of these complexes on cells may be rotationally immobile. A comparison of the initial phosphorescence anisotropy values for erythrosin-labeled IgE-receptor complexes on cells and vesicles reveals a fast component of rotational motion that is greater on the vesicles and may be due to a site of segmental flexibility in the receptor itself. Dimers of IgE-receptor complexes formed with anti-IgE monoclonal antibodies appear to be largely immobile on cells, but they are mobile on vesicles with a 2-fold larger rotational correlation time than the monomeric complexes. The results suggest that dimeric IgE-receptor complexes undergo interactions with other membrane components on intact cells that do not occur on the membrane vesicles. The possible significance of these interactions to receptor function is discussed.

Animals↗
PubMed1991

Conformations of IgE bound to its receptor Fc epsilon RI and in solution.

Previous resonance energy transfer studies suggested that murine immunoglobulin E (IgE) is bent near the junction of its Fc and Fab segments when bound to its high-affinity receptor (Fc epsilon RI) on RBL cells. To examine further the conformations of IgE, both bound to this receptor and in solution, a mutant recombinant IgE (epsilon/C gamma 3*) was prepared that has a cysteine replacing a serine near the C-terminal ends of the heavy chain. The introduced cysteine residues provide a means for specific modification of IgE, and the sulfhydryl groups were selectively labeled with fluorescein-5-maleimide (FM-epsilon/C gamma 3*). This IgE also binds a 5-(dimethylamino)naphthalene-1-sulfonyl (DNS) group in the antigen-binding sites. Resonance energy transfer experiments carried out on receptor-bound FM-epsilon/C gamma 3* yielded a distance of 53 A between fluorescein near the C-terminal end of the Fc segment and amphipathic acceptor probes at the membrane surface. The average distance between this C-terminal fluorescein and acceptor eosin-DNS in the antigen-binding sites at the N-terminal ends of the Fab segments was found to be 69 A. These results combine with those from previous structural studies to provide an unprecedented detailed description of the bent geometry of IgE bound to its receptor on the membrane. Energy transfer measured for FM-epsilon/C gamma 3* in solution between fluorescein near the C-terminal end of the Fc segment and eosin-DNS at the N-terminal ends of the Fab segments indicates that the average distance between these probes is about 71 A.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Characterization of increased K+ permeability associated with the stimulation of receptors for immunoglobulin E on rat basophilic leukemia cells.

Aggregation of immunoglobulin E-receptor complexes on the surface of rat basophilic leukemia cells stimulates an increase in plasma membrane K+ permeability that is monitored as an increase in the rate of efflux of preloaded 86Rb+. A major component of this stimulated 86Rb+ efflux appears to be due to a Ca(2+)-activated K+ channel because it is inhibited by quinidine in parallel with the inhibition of degranulation and membrane potential repolarization, it is blocked by 0.1 mM La3+, and it is dependent on external Ca2+. Depolarization of the plasma membrane by carbonyl cyanide 3-chlorophenylhydrazone inhibits stimulated Ca2+ influx and prevents antigen-induced 86Rb+ efflux, and increased external Ca2+ partially restores 86Rb+ efflux under these conditions. In addition, potentiation of antigen-stimulated Ca2+ influx by pretreatment with cholera toxin increases the initial rate of stimulated 86Rb+ efflux. Another component of antigen-stimulated K+ efflux appears to be mediated by a guanine nucleotide-binding protein because pretreatment of rat basophilic leukemia cells with pertussis toxin decreases the initial rate of antigen-stimulated 86Rb+ efflux to 40% of that for the untreated cells. Stimulated 86Rb+ efflux is also observed when ionomycin is used to increase cytoplasmic Ca2+ and to trigger membrane depolarization. The efflux stimulated by ionomycin is inhibited by quinidine but not by pertussis toxin pretreatment; thus, it appears to occur through the Ca(2+)-activated K+ efflux pathway. It is proposed that these K+ efflux pathways serve to sustain the Ca2+ influx that is necessary for receptor-mediated triggering of cellular degranulation.

Animals↗
PubMed1991

Dissociation kinetics of bivalent ligand-immunoglobulin E aggregates in solution.

We study the dissociation of preformed bivalent ligand-bivalent receptor aggregates in solution, where the ligand is a symmetric bivalent hapten with two identical 2,4-dinitrophenyl (DNP) groups and the receptor is a fluorescein-labeled monoclonal anti-DNP IgE. We promote dissociation in two ways: by the addition of high concentrations of a monovalent hapten that competes for IgE binding sites with the bivalent hapten and by the addition of high concentrations of unlabeled IgE that binds almost all ligand binding sites that dissociate from labeled IgE. We investigate both theoretically and experimentally the two types of dissociation and find them to be quite different. Theory predicts that their kinetics will depend differently on the fundamental rate constants that characterize binding and aggregation. Using monovalent ligand to promote dissociation, we find that the fraction of labeled IgE sites bound to bivalent ligand decays with a slow and fast component. The fast decay corresponds to the dissociation of a singly bound DNP hapten. The interpretation of the slow decay depends on the detailed way in which ligand-receptor aggregates break up. We show that one possible explanation of these data is that small stable rings form before the addition of monovalent ligand. Other possible explanations are also presented.

Animals↗
PubMed1991

Bivalent ligand dissociation kinetics from receptor-bound immunoglobulin E: evidence for a time-dependent increase in ligand rebinding at the cell surface.

The bivalent ligand N,N'-bis[[epsilon-[(2,4- dinitrophenyl)amino]caproyl]-L-tyrosyl]cystine [(DCT)2-Cys] binds and cross-links anti-dinitrophenyl (DNP) immunoglobulin E (IgE)-receptor complexes on the cell surface of rat basophilic leukemia cells. The rate of dissociation of this bound ligand was monitored by using a fluorescence method under two different conditions. In one case the monovalent ligand DCT was added in large excess to prevent the dissociating ligand from rebinding to unoccupied antibody combining sites. Under these conditions, dissociation of the bivalent ligand from IgE-sensitized cells proceeds to completion with kinetics that are well described by two rate constants that are independent of the time of preincubation of the bivalent ligand with the cells. In the second case, dissociation of (DCT)2-Cys from cell-bound anti-DNP IgE was monitored in the presence of a large excess of anti-DNP IgE in solution that acts as a sink to absorb the dissociated ligand. Under these conditions, the bivalent ligand becomes more resistant to dissociation as the preincubation time of the bivalent ligand with the cells is increased. An increasing fraction of the bound ligand does not dissociate on a measurable time scale in the presence of this sink. The results indicate that cell-associated IgE-receptor complexes undergo a time-dependent change that facilitates the reformation of the cross-linked state when one end of the ligand dissociates to break up the existing cross-link. The possible physical basis and functional implications of these results are discussed.

Animals↗
PubMed1991

Characterization of new rat anti-mouse IgE monoclonals and their use along with chimeric IgE to further define the site that interacts with Fc epsilon RII and Fc epsilon RI.

Three rat monoclonal antibodies specific for mouse IgE (C12B9, 23G3, and B1E3) were established by using monoclonal anti-DNP mouse IgE (mIgE) as immunogen. These antibodies, as well as a fourth, (R1E4) were characterized. It was found that one antibody (C12B9) recognizes an allotypic determinant (Igh-7a) found on the C epsilon chain of mIgE. Antibody cross-blocking studies and epitope mapping studies using recombinant mIgE indicated that 3 antibodies (C12B9, R1E4 and 23G3) were directed against the C epsilon 3 domain while one (B1E3) was directed against the C epsilon 4 domain. A highly specific sandwich RIA for mIgE was developed using these antibodies. Use of these monoclonal anti-mIgE antibodies in conjunction with recombinant chimeric mIgE-human IgG1 molecules, demonstrated that the C epsilon 3 domain is important in the binding of mIgE to the murine B cell Fc epsilon RII as well as to the murine mast cell F epsilon RI. The presence of the C epsilon 4 domain influenced the binding of the recombinant IgE to the Fc epsilon RII; in contrast to the C epsilon 4 domain had no effect on binding to the Fc epsilon RI.

Animals↗
PubMed1990

Recombinant human IgG1-murine IgE chimeric Ig. Construction, expression, and binding to human Fc gamma receptors.

We have constructed a set of chimeric Ig by exchanging corresponding H chain C domains between human (hu) IgG1 and murine (m) IgE. We used this set of Ig to dissect the interaction of individual Ig domains with human Fc gamma receptors. Only one of the chimeras, epsilon/C gamma 2,3 (an mIgE with C epsilon 3 and C epsilon 4 replaced by C gamma 2 and C gamma 3 from huIgG1), binds tightly to the human Fc gamma RI on U937 cells. We found that epsilon/C gamma 2,3 has only twofold lower affinity for Fc gamma RI as compared to huIgG1. The gamma/C epsilon 4 (huIgG1 with C epsilon 4 replacing C gamma 3) binds weakly to Fc gamma RI. The other chimeric Ig, epsilon/C gamma 3, epsilon/C gamma 2, and gamma/C epsilon 3, as well as mIgE do not bind detectably to Fc gamma RI. From these data we conclude that the C gamma 2 domain is crucial for binding and contains the majority of the binding site for Fc gamma RI on IgG1. The C gamma 3 domain makes a smaller contribution to the binding, and the C gamma 1 domain and the hinge region have very little effect on the Fc gamma RI-IgG1 interaction. The chimeric epsilon/C gamma 2,3 and huIgG1 both mediate the formation of rosettes between K562 cells and antigen-sensitized E with similar concentration dependences. These results suggest similar ability to bind to Fc gamma RII. The other chimeric Ig do not cause rosettes in this assay system. Hence, both C gamma 2 and C gamma 3 seem to be required for binding to Fc gamma RII, but the C gamma 1-hinge region has no detectable effect.

Animals↗
PubMed1990

Mapping the site of interaction between murine IgE and its high affinity receptor with chimeric Ig.

We have investigated the interaction of mouse (m) IgE with its Fc epsilon RI on rat basophilic leukemia cells using a set of chimeric Ig that were constructed by exchanging homologous H chain C domains between human (hu) IgG1 and mIgE. Binding affinities were examined with equilibrium and kinetic measurements, and we found that epsilon/C gamma 3 (mIgE with C epsilon 4 replaced by C gamma 3) was indistinguishable from mIgE. The huIgG1 and the other chimeric Ig, which did not contain both C epsilon 2 and C epsilon 3, did not bind detectably to rat basophilic leukemia cells (Ka less than 10(6) M-1). The ability of these chimeric Ig to stimulate a cellular response (degranulation) in the presence of multivalent Ag was also tested. The epsilon/C gamma 3 was indistinguishable from mIgE in eliciting a high level of degranulation, whereas the other chimeric Ig stimulated no response even when they were preaggregated to enhance their binding avidity. These results demonstrate that C epsilon 4 may be replaced by C gamma 3 without affecting the binding and cell activating properties of mIgE. The lack of binding by the other chimeric Ig indicates that both C epsilon 2 and C epsilon 3 are necessary for the binding interaction.

Animals↗
PubMed1990

Microfilaments regulate the rate of exocytosis in rat basophilic leukemia cells.

Disruption of microfilaments in rat basophilic leukemia (RBL) cells by exposure to cytochalasin B is observed to potentiate the rate of antigen-stimulated secretion from these cells. Under these conditions, cytochalasin B is without effect on the antigen-stimulated production of inositol phosphates or 45Ca2(+)-influx. In streptolysin-O-permeabilized RBL cells, cytochalasin B is observed to potentiate the rate of secretion in response both to guanosine 5'-(2-thio)-O-triphosphate (GTP gamma S) and to Ca2+ (buffered between 0.1 and 10 microM). However, under these conditions, cytochalasin B does not affect to antigen-stimulated production of inositol phosphates. Consistent with these data, microfilaments are proposed to regulate a terminal step in exocytosis, in a physiologically relevant manner.

Actin Cytoskeleton↗
PubMed1990

A nanosecond fluorescence depolarization study on the segmental flexibility of receptor-bound immunoglobulin E.

Time-resolved fluorescence anisotropy measurements have been used to examine the segmental flexibility of anti-dansyl immunoglobulin E (IgE) bound to its high-affinity receptor on membrane vesicles from rat basophilic leukemia cells. Although IgE in this complex exhibits only a restricted angular range of segmental motion, much of this restricted motion occurs on a relatively rapid time scale. A fast component of motion with a rotational correlation time of 15-35 ns may correspond to the twisting of Fab segments about their major axis. Intermolecular cross-linking by a short bivalent ligand, N,N'-didansylcadaverine, results in complete loss of this segmental motion. Solubilization of monomeric IgE-receptor complexes using a zwitterionic detergent results in a time-dependent anisotropy decay that exhibits both a fast component and a slower component that is intermediate between the decay for soluble and membrane-bound forms of IgE at long times after excitation. These results are discussed in terms of a model in which binding of IgE to its membrane-bound receptor restricts not only its global rotation but also its slower modes of segmental flexibility as well, while allowing its Fab segments to undergo rapid reorientation within a limited angular range.

Animals↗
PubMed1990

A guanine nucleotide-binding protein participates in IgE receptor-mediated activation of endogenous and reconstituted phospholipase A2 in a permeabilized cell system.

Activation of phospholipase A2 (PLA2) by the aggregation of receptors for immunoglobulin E (IgE) can be studied in streptolysin O-permeabilized rat basophilic leukemia cells. Under these conditions, 40 microM guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) stimulates PLA2 activity 5-6-fold when free Ca2+ concentrations are buffered at 10(-7)-10(-5) M. Antigen-mediated cross-linking of receptors for IgE synergizes with low concentrations of GTP gamma S (0.1 microM) to cause similar stimulation. When the endogenous PLA2 activity is inactivated by chemical modification, we find that exogenously supplied PLA2 from porcine pancreas and Naja naja venom is also activated by the aggregation of cell-surface IgE receptors in these permeabilized cells. As with endogenous PLA2, GTP gamma S synergizes with IgE receptor-aggregation to activate exogenous PLA2 approximately 10-fold at 10(-7)-10(-6) M free Ca2+. These data indicate that receptor-mediated activation of a guanine nucleotide-binding protein can shift the Ca2+ dependence of PLA2 activity resulting in greatly enhanced activity at physiological concentrations of intracellular free Ca2+. The partial reconstitution of various PLA2 forms into such a broken-cell system offers a new approach for studying the mechanisms of G-protein-mediated activation of PLA2.

Animals↗
PubMed1990

Characterization of model antigens composed of biotinylated haptens bound to avidin.

Model antigens of defined structure and limited valency have been prepared by binding 2,4-dinitrophenyl (DNP)-biotin haptens of different lengths to the biotin binding sites of avidin. These complexes have been characterized structurally by spectroscopic methods and functionally by testing their ability to crosslink cell-bound anti-DNP immunoglobulin E-receptor complexes to stimulate degranulation of rat basophilic leukemia cells. One of these haptens, 1-DNP-amino-12-biotinamido dodecane, is shown to have only two tight binding sites per avidin molecule, and the resulting bivalent (DNP-biotin)-avidin antigen is found to stimulate substantial degranulation. Another DNP-biotin hapten that is approximately 10 A longer has four tight binding sites per avidin and, when bound to avidin, has greater activity similar to a highly DNP-conjugated multivalent antigen. These and other (DNP-biotin)-avidin complexes described here represent a new group of chemically well-defined antigens that are useful for studying the binding to and functional crosslinking of cell-bound immunoglobulins in immunological responses.

Animals↗
PubMed1989

Bivalent binding of an anti-CD3 antibody to Jurkat cells induces association of the T cell receptor complex with the cytoskeleton.

Ligand binding and cross-linking of TCR/CD3 complex leads to T cell stimulation in immunologic responses. As a prelude to investigating the dynamic interactions of these receptors, we have characterized binding of the mAb OKT3 specific for CD3 on Jurkat cells. The association of both OKT3 and its Fab' fragment is rapid at 4 degrees C, and dissociation of Fab' is also rapid, but dissociation of OKT3 is slow, indicating bivalent binding in this case. Dissociation of OKT3 is substantially accelerated at 37 degrees C if internalization is prevented. From the concentration dependence, the binding of OKT3 at 4 degrees C appears to be very tight whereas binding of the Fab' fragment is weaker and biphasic. Scatchard analysis of the Fab' equilibrium binding data indicates two binding sites with KD values of 5.1 x 10(-9) M and 2.7 x 10(-8) M. The very tight binding of the bivalent antibody may be caused by inter- or intramolecular cross-linking between these sites. If Jurkat cells are warmed to 37 degrees C, there is an energy-dependent increase by about one-third of sites bound by OKT3 or its Fab' fragment over that seen at 4 degrees C. This increase may be related to a receptor recycling process because internalization of a similar number of the bound ligands occurs at similar rates. Other experiments have revealed that OKT3, but not its Fab' fragment, causes the receptor complex to become associated with the detergent-insoluble cytoskeleton, and there are also insoluble intracellular OKT3-binding sites. These cross-linking-induced receptor-cytoskeletal interactions are sensitive to moderate changes in salt concentration that should allow their molecular basis to be investigated.

Antibodies, Monoclonal↗
PubMed1989

Antigen-triggered membrane potential changes in IgE-sensitized rat basophilic leukemia cells: evidence for a repolarizing response that is important in the stimulation of cellular degranulation.

We have studied Ag-induced membrane potential changes of rat basophilic leukemia cells by using the potential-sensitive dye, bis-(1,3-diethylthiobarbiturate)trimethineoxonol. A rapid membrane depolarization is triggered by a multivalent Ag, and it has a bell-shaped dose dependence that parallels the degranulation response but not the extent of cross-linking of the IgE-receptor complexes. As the temperature is reduced from 37 degrees C, this depolarization response slows and decreases in magnitude until complete inhibition is observed at 15 degrees C, similar to the temperature dependence previously observed for the Ag-stimulated rise in cytoplasmic Ca2+ and for degranulation. The results imply that a highly temperature-dependent step subsequent to Ag binding and cross-linking is necessary for the depolarization response. A partial return to the resting potential is seen to follow the depolarization response to Ag. This repolarization process is inhibited by quinidine.HCl and Ba2+ in parallel with an inhibition of the degranulation response. Repolarization is not affected by 4-aminopyridine or by the absence of K+ in the external buffer. These data suggest that the repolarization is caused by a previously uncharacterized K+ channel.

Animals↗