Monovalent Ligands Research Articles

Increased affinity of dimeric enkephalins is not dependent on receptor density

The equilibrium binding and dissociation kinetics of the enkephalin dimer bis-(D-Ala 2-D-Leu 5-enkephalin)-ethylenediamide (designated DPE 2) to neuroblastoma glioma NG108-15 cells were investigated and compared with the monomers D-Ala 2-D-Leu 5-enkephalin (DADL) and D-Ala 2-Leu 5-enkephalinamide (DALEA). Binding was studied after exposure of the membrane to increasing concentrations of the irreversible delta receptor selective ligand FIT in order to decrease the density of binding sites on the cell membrane. The increased affinity of DPE 2 did not revert to that of the monomer DADL by this reduction of binding sites. Similarly, the dissociation of DPE 2 did not approach that of the monomer DALEA in the presence of 1 μM DALEA. These data strongly suggest that crosslinking does not occur, and fail to confirm the hypothesis that dimers with short spanning chain length aid the clustering of receptors. We postulate: 1) If the dimer binds to a bivalent binding site, the monovalent binding state of our bivalent ligand may not exist to an appreciable extent, and 2) the bivalent ligand cannot bind when the binding site is irreversibly blocked by a monovalent ligand.

Virus-receptor interaction in the adenovirus system: characterization of the positive cooperative binding of virions on HeLa cells.

The established positive cooperativity of adenovirus 2 binding to HeLa cells revealed a strong temperature dependence. The degree of cooperativity, quantified by means of Hill coefficients, progressively increased from 10 degrees C to reach a maximum level, which was maintained between 20 and 37 degrees C. On the other hand, negative cooperativity of virion attachment was apparent at 3.0 degrees C and on glutaraldehyde-stabilized cells. The corresponding monovalent ligand of the system, the fiber antigen, demonstrated only weak-positive cooperativity of the binding at 37.0 degrees C, which was absent at 3.0 degrees C. Dithiothreitol and dansylcadaverine, reagents inhibiting clustering of ligand-receptor complexes in the plasma membrane, markedly reduced the degree of positive cooperative binding at 37.0 degrees C. Evidently, the positive cooperative binding of adenovirus to HeLa cells at 37.0 degrees C is a consequence of both the multivalency of virus attachment proteins, i.e., fibers, on the virion and of the capacity of the receptor sites to migrate in the plane of the plasma membrane, forming local aggregates of virus-receptor site complexes.

Endocytosis of the membrane immunoglobulins of mouse spleen B-cells: a quantitative study of its rate, amount and sensitivity to physiological, physical and cross-linking agents.

A quantitative analysis by flow cytometry of the rate and extent of endocytosis of ligands bound to the membrane immunoglobulins of mouse B-splenocytes is reported. The temperature dependence and the response to inhibitors of oxidative metabolism are described. Inhibitors of the cytoskeleton (cytochalasin B, vinblastine and colchicine) and of calmodulin (trifluoperazine) do not interfere with endocytosis at non-lethal doses. Similarly fetal calf serum does not modify the rate and extent of the process. Endocytosis occurs in a similar time range, but to a lesser extent, when the ligand is monovalent than when it cross-links the membrane immunoglobulins. Finally, it is shown that, within minutes after its internalization, the divalent ligand is found in an acidic environment, while the monovalent ligand is not. These results, in agreement with the model of receptor recycling, suggest that the divalent ligand-receptor complex is indeed internalized and captured in an acidic environment while the monovalent ligand-receptor complex is internalized and probably rapidly recycled back to the cell surface.

The increase in intracellular free calcium associated with IgG gamma 2b/gamma 1 Fc receptor-ligand interactions: role in phagocytosis.

The concentration of cytosolic free calcium, [Ca2+]i, was measured in J774 mouse macrophages by use of the fluorescent indicator quin-2. Resting [Ca2+]i was 87 nM. Addition of a number of specific ligands to the immunoglobulin gamma 2b/gamma 1 Fc receptor resulted in a transient increase in [Ca2+]i, the magnitude of which depended on the extent of receptor aggregation. Monovalent ligands gave only a small Ca2+ signal but blocked cell response to subsequent addition of multivalent ligands. Incubation with antibody-coated erythrocytes raised macrophage [Ca2+]i to micromolar levels. [Ca2+]i changes were only partially inhibited by the absence of external Ca2+, suggesting the release of Ca2+ from internal stores in addition to an influx of external Ca2+. These internal stores were not limited to mitochondria. An optimal range of [Ca2+]i was required for phagocytosis. Buffering [Ca2+]i with quin-2 and treating cells with quinine in the absence of external Ca2+ resulted in inhibition of phagocytosis. Increasing [Ca2+]i to micromolar levels with the calcium ionophore A23187 also resulted in similar inhibitory effects. We suggest the involvement of localized cytosolic Ca2+ gradients in generating the signals necessary for phagocytosis.

Internalization and degradation of macrophage Fc receptors bound to polyvalent immune complexes.

We have studied the Fc receptor-mediated pinocytosis of immunoglobulin G (IgG)-containing immune complexes by mouse macrophages. IgG complexes were formed from affinity-purified rabbit dinitrophenyl IgG and dinitrophenyl modified BSA at molar ratios of 2.5-10:1. Both the specificity of binding and the fate of internalized receptors were analyzed using monoclonal and polyclonal anti-Fc receptor antibodies. Based on the susceptibility of surface-bound ligand to release by proteolysis, we have found that at 37 degrees C, 125I-labeled IgG complexes were rapidly internalized (t1/2 less than 2 min) and delivered to lysosomes; acid-soluble 125I was detectable in the growth medium within 5-10 min of uptake. However, kinetic evidence indicated that Fc receptors were not efficiently re-used for multiple rounds of ligand uptake. Instead, macrophages that were exposed continuously to saturating concentrations of IgG complexes exhibited a selective and largely irreversible removal of Fc receptors from the plasma membrane. This loss of surface receptors correlated with an increased rate of receptor turnover, determined by immune precipitation of Fc receptors from 125I-labeled macrophages. Thus, in contrast to the results obtained in the accompanying paper (I. Mellman, H. Plutner, and P. Ukkonen, 1984, J. Cell Biol. 98:1163-1169) using a monovalent ligand, these data indicate that the interaction of Fc receptors with polyvalent complexes leads to the degradation of both ligand and receptor following their delivery to lysosomes.

Are Fc gamma receptors naturally clustered on normal human B lymphocytes?

The Fc gamma receptor (FcR) on normal human B lymphocytes was studied by using electron-microscopic monovalent ligands made of one ferritin (Fer) and one rabbit IgG anti-Fer. The binding of ligands was ascertained with the modified Coombs mixed antiglobulin reaction. The receptors were found clustered at 0 degree C, which was not affected even by examining the coated cells without washing away excess of the ligand solution, or by cross-linking bound ligands with a second antibody. The clusters varied in length, measuring up to 220 nm. Most of them were less than 110 nm long, and were seen as spots. When lymphocytes were preincubated with both cytochalasin B and colchicine, the clustering of FcR was not affected. Our findings show that FcR on B lymphocytes are naturally aggregated, which is not primarily mediated with the cytoskeletons made of actin microfilaments and microtubules.

The role of non-immune IgG in controlling IgG-mediated effector functions

The majority of evidence supports the conclusion that IgG-dependent effectors respond to antibodies which have been polymerized artifically or by polyvalent antigens, but not to monomeric IgG antibodies. Effectors can distinguish polymerized IgG antibodies from monomeric IgG because they contain multiple receptor units and can interact multivalently with polymerized IgG. However, monomeric IgG is present at very high concns in plasma and interstitial fluids and will inhibit multivalent interactions in vivo between polymerized antibody and effectors. Such inhibition raises the question of how IgG-mediated effector responses could function in vivo. In this review we present a mathematical model which quantitatively predicts how polyvalent ligands interact multivalently with receptors in the presence of excess monovalent ligand. We then show that results from experiments in vitro using such diverse systems as the binding and endocytosis of immune complexes by macrophages, complement-mediated lysis of antibody-coated target cells, and ADCC can be explained qualitatively by the model. We conclude that monomeric IgG does not totally inhibit IgG-mediated effector functions but, rather, raises the threshold of antibody binding which is required to elicit a response. We then consider how non-immune IgG may serve as a homeostatic regulator of IgG-dependent responses, in vivo, perhaps for the purpose of inhibiting responses to low levels of cell-bound IgG autoantibodies.

Macrophage membrane potential changes associated with gamma 2b/gamma 1 Fc receptor-ligand binding.

We have studied the effects of specific ligands of the receptor for the IgG Fc fragment (FcR) on the membrane potential (delta psi) of the macrophage cell line J774 by the [3H]tetraphenylphosphonium ion equilibration technique. We observe a membrane depolarization with binding of FcR ligands that is dependent on the degree of receptor crosslinking. Binding of the FcR by monovalent ligands is not sufficient to induce a significant drop in delta psi, but a sustained depolarization lasting approximately equal to 20 min occurs with insoluble multivalent ligands. This FcR-mediated depolarization can be inhibited by substitution of Na+ from the cell incubation medium with monovalent choline cation, indicating that depolarization is due to Na+ influx into the cell. The extracellular Ca2+ does not play a significant role in membrane depolarization. The depolarization response is not triggered by monoclonal antibodies directed against three other major macrophage surface antigens. The cell depolarization mediated by FcR ligands is followed by a prolonged hyperpolarization that can be partially blocked by ouabain and quinine, indicating that the hyperpolarization response is a result of a combination of a Na+,K+-ATPase activity and a Ca2+-activated K+ conductance. These data support our hypothesis that the mouse macrophage IgG FcR is a ligand-dependent ion channel.

Coordination compounds of heterocyclic AZO derivatives II‐transition metal chelates of some pyrazolone AZO dyes

AbstractThe chelates of some 4‐arylazo‐3‐amino‐pyrazolone derivatives with Mn(II), Fe(III), Zr(IV), Pd(II) and Ag(I) are prepared and characterized. It is concluded that the ligands and the complexes exist mainly in the hydrazone‐keto form. It is confirmed that such compounds are coordinated to the metal ion as monovalent bidentate ligands, L−, and the bonding sites are the oxygen of the carbonyl group and the α‐nitrogen of the arylazo group.

Mono‐and bi‐nuclear metal complexes of the Schiff bases, arylidenes‐anthranilic acid

AbstractSchiff bases, arylidenes‐anthranilic acid complexes with Th(IV), UO2(II), La(III), Ce(III) and Zr(IV) have been isolated. The structure of the complexes is recognized by elemental analysis, electronic spectra and i.r. spectral data as well as conductance measurements. The salicylidene‐anthranilic acid L3 is coordinated to the metal ion as bivalent tridentate ONO ligand (L−2) while the other compounds studied are coordinated to the metal ion as monovalent bidentate ON ligand. The molecular structure effect of these compounds on the strength of their complexes is discussed.It is identified that the Cu(II) complex of L3 can act as bidentate ligand, coordinating through its cis oxygen atoms to form binuclear metal complexes.

7] Role of diffusion regulation in receptor-ligand interactions

Publisher Summary This chapter describes the way in which the physical environment of a receptor influences its interaction with a ligand and the way the effects of that influence can be quantitatively characterized. It considers the influence of two properties of a cell: its size and the fluidity of its surface. It also describes the theoretical considerations indicating that, when reactions are diffusion controlled, the kinetics of solution phase ligand interacting with receptors depends markedly on whether the receptor is dispersed or cell bound. This difference is a natural consequence of Brownian movement in the vicinity of the cell. By contrast, the equilibrium constant for specific binding is more or less independent of whether the receptor is cell bound, unless constraints imposed by membrane components influence its conformation. The simultaneous determination of equilibrium constants for the cell-bound and the soluble receptor is, therefore, a useful indicator of conformation. Methods for obtaining them by binding and response inhibition are also reviewed in the chapter. The development is restricted to spherical cells with uniformly distributed receptors. The chapter also discusses the theory of attachment and inhibition of monovalent and bivalent ligands to receptors on fluid and static surfaces.

Narcotic antagonistic potency of bivalent ligands which contain beta-naltrexamine. Evidence for bridging between proximal recognition sites.

Two-bivalent ligands (P-X-P) containing the beta-naltrexamine pharmacophore (P) and a connecting oligoethylene glycol spanner (X) were synthesized and evaluated for narcotic antagonistic activity in the guinea pig ileum (GPI) and mouse vas deferens (MVD). The bivalent ligand 2 whose spanner contains three ethylene units possessed 10-fold greater antagonistic potency than its monovalent analogue (4) in antagonizing the effects of ethylketazocine (EK) on the GPI, while no differential antagonism of morphine was observed among the compounds. In the MVD, 2 was not substantially more potent than 4 as an antagonist against [D-Ala2,D-Leu5]enkephalin (DADLE). The bivalent ligand 3, whose spanner contains six ethylene units, exhibited 15 times greater potency in antagonizing the agonist effects of DADLE on the MVD than its monovalent ligand 4. No marked increase in the ability of 3 to antagonize the effects of morphine or EK on the GPI was observed. The data indicate that mu, kappa, and delta opioid receptors exhibit different selectivity toward bivalent ligands whose spanner lengths differ. The enhanced potency associated with different receptor interactions is consistent with simultaneous occupation of proximal recognition sites. Whether such proximal recognition sites are identical or different remains to be clarified. The distance between proximal sites appears to depend on the opioid receptor subtype involved.

Characterization of lipoteichoic acid binding to polymorphonuclear leukocytes of human blood.

Human polymorphonuclear leukocytes (PMN) were shown to possess specific binding sites for lipoteichoic acid (LTA). LTA binding was reversible and time and temperature dependent. Scatchard plot analysis revealed an apparently single population of 6.6 X 10(6) LTA binding sites per PMN with a dissociation constant of 5.6 microM. Attachment of an avirulent, unencapsulated, M-negative strain of group A streptococci to PMN was inhibited by LTA, but not by other bacterial somatic antigens tested. Occupation of 30% of the LTA binding sites resulted in greater than 70% inhibition of streptococcal attachment to PMN. In contrast, LTA failed to block attachment of Escherichia coli or antibody-coated streptococci, indicating that binding sites for E. coli and the Fc portion of immunoglobulin G are distinct from those for LTA. Immunofluorescent studies demonstrated that LTA remained uniformly bound to PMN membranes for as long as 2 h at 37 degrees C. Cross-linking of PMN-bound LTA with anti-LTA resulted in rapid capping of LTA receptor sites. The results suggest that LTA is a monovalent ligand interacting with mobile receptors in the plasma membrane of PMN.

Internalization of α2 macroglobulin in receptosomes. Studies with monovalent electron microscopic markers

Previous studies using multivalent, peroxidase-labeled antibody for localization of α 2 M have demonstrated that the binding of α 2-macroglobulin ( α 2 M) to randomly distributed receptors on the surfaces of fibroblasts initiates the accumulation of α 2 M-receptor complexes in clathrin-coated pits. The α 2 M-receptor complexes are then internalized into a specialized vesicle termed the receptosome. In the present study we have used three different monovalent ligands to localize α 2 M and show that the endocytosis of α 2 M-receptor complexes by the receptosomal pathway is not initiated as a result of antibody-induced cross-linking of the α 2 M-receptor complexes. To perform these studies the following monovalent markers of α 2 M were prepared for electron microscopic visualization: (1) a monovalent hybrid antibody directed against α 2 M and ferritin; (2) a monovalent hybrid antibody directed against α 2 M-peroxidase; and (3) a direct 1:1 conjugate of α 2 M-peroxidase. We find that all three of the markers are internalized by the ligand pathway previously described using multivalent labels. The steps involved are clustering of α 2 M receptor complexes in coated regions of the plasma membrane followed by endocytosis of α 2 M into receptosomes. Our results are contrasted with previous studies on lymphocytes in which antibody induced cross-linking of membrane antigens was necessary for triggering their pinocytosis. The methods described in this paper are applicable for visualizing at the electron microscopic level the internalization of other ligands and hormones.

Increase of conformational stability of homogeneous rabbit immunoglobulin g after hapten binding

Hydrogen-deuterium exchange, adiabatic scanning microcalorimetry and difference sedimentation were used to detect ligand-induced conformational changes in a homogeneous rabbit antibody. Site-filling monovalent and bivalent ologosaccharide ligands, derived from the type III pneumococcal polysaccharide, were used. Only those antigen-antibody complexes which were monomeric with respect to antibody were included into this study. Hydrogen-deuterium exchange reflects the probability of solvent exposure of peptide hydrogens. In this study, the comparative analysis of the probability distributions for various antibody-ligand complexes demonstrates that most if not all of the observable exchange rates were affected by the bincling of hapten. Scanning microcalorimetry detects changes in the overall conformational stability of the antibody molecule. Biphasic heat absorption curves were obtained, reflecting biphasic heat-induced conformational transitions. The significant hapten-induced shift in the temperature of the first transition was interpreted as a consequence of tightening in the domain-domain interactions, while the slight shift in the second transition can be explained by a stabilization of the domains themselves. No difference could be detected in the hydrodynamic properties of the liganded and the unliganded antibodies by difference sedimentation. The effect of monovalent or bivalent hapten bincling upon antibody conformation manifests itself in restricted conformational motility and enhanced conformational stability, without any detectable change in the shape or the volume of the molecule.

Surface distribution and endocytosis of the NS3 antigen of neuroblastoma: An immunocytochemical study

The surface distribution and adsorptive endocytosis of the NS3 antigen of cultured murine neuroblastoma cells has been studied by indirect immunoperoxidase and immunofluorescent techniques. We have found that 1. 1. The NS3 antigen is present on plasma membranes of perikarya and processes and of murine neuroblastoma 2. 2. Surface redistribution of NS3 induced by anti-NS3 sera occurred in several ‘patches’ rather than in a single ‘cap’; we suggest that asymmetric cells exposed to a bivalent ligand show multiple foci of segregation of plasma membrane antigens. 3. 3. NS3 antigen was observed at the cleavage furrow of mitotic cells at anaphase or telophase 4. 4. Neoplastic neurons are endowed with a system of cisternae and vesicles which is analogous to GERL (Golgi-Endoplasmic Reticulum-Lysosome) of normal neurons 5. 5. Anti-NS3 antibodies linked by bivalent ligands undergo endocytosis into putative lysosomes, while anti-NS3 linked by monovalent ligands undergo endocytosis into GERL.

Does lectin-receptor complex formation produce zones of restricted mobility within the membrane?

IT is well established that component lipids and proteins are mobile in the plane of the membrane1,2. This lateral mobility could have an important role in the transmission of signals through the membrane. For example, there is much evidence that aggregation of receptors induced by divalent antibodies or by chemical cross-linking can reproduce the biological action of the corresponding ligands3–5. It was shown recently that receptors for monovalent ligands like insulin and epidermal growth factor aggregate into patches on the cell surface after interaction with the corresponding hormone. Clustered receptors accumulate at coated regions of the membrane and become internalised in endocytic vesicles6,7. Aggregation is dependent on receptor occupancy by the active ligand7. Here we show that lateral mobility is also dependent on receptor occupancy. Our approach has been to compare the lateral mobilities of free and ligand-bound receptors using cultured cells placed in a steady electric field. This permits the measurement of lateral long-range (over the whole cell) mobility of the receptors with and without the corresponding ligands8. We have compared this method with the dynamic method of fluorescence photobleaching recovery (FPR) which only permits measurement of the mobility of fluorescent ligand-receptor complexes in the range of the size of the laser beam diameter (∼ 3 μm)9–12. We have studied the lateral mobility of concanavalin A (Con A) and ricin receptors from non-malignant fibroblasts 3T3 and malignant neuroblastoma NS-20 and C6 glial cells with both techniques. Our results suggest that lectin-receptor formation results in the production of zones of restricted mobility in the membrane.

(Na+ + K+)-adenosine triphosphatase of mammalian brain. Catalytic and regulatory K+ sites distinguishable by selectivity for Li+.

Li+, K+, and Rb+ are compared as activators of the hydrolysis of p-nitrophenylphosphate by beef brain (Na+ + K+)-ATPase. Previous experiments have established two classes of K+ binding sites that are involved in this reaction: "catalytic sites" have the higher affinity, their occupation is essential for catalytic activity, and they appear to correspond to the extracellular binding sites for active K+ transport; regulatory sites appear to have an allosteric function to "unmask" the catalytic sites. A separate set of Na+-binding regulatory sites bring about a similar unmasking of catalytic sites under phosphorylating conditions. Rb+ can activate p-nitrophenylphosphate hydrolysis both in the presence and absence of Na+ and, thus, can interact effectively with both K+ regulatory and catalytic sites. Li+ does not activate p-nitrophenylphosphate hydrolysis at 25 degrees C in the absence of other monovalent ligands. Li+ does activate when the catalytic sites are exposed by Na+ + ATP. Thus, K+ regulatory and catalytic sites differ in their cation selectivity. At temperatures less than 25 degrees C Li+ is able to activate the phosphatase reaction in the absence of other monovalent ligands: maximum activity occurs at 10-12 degrees C. A plot of the ratio, Li+ activation/K+ activation, as a function of temperature shows that the allosteric transition that unmasks catalytic sites occurs spontaneously with decreasing temperatures.

Interaction of divalent antibody with cell surface antigens

This paper presents a thermodynamic description of the interaction between divalent protein in solution and monovalent ligand attached to a cell membrane, but freely mobile therein. The intrinsic differences between this system and interaction which takes place in a uniform bulk solution are discussed. Specific problems which are addressed include the determination of the number of ligand sites on a specific cell system, comparison of several cell systems in terms of common antigenic components, comparison of different antibody preparations, and attempts to study solubilized cell-surface antigens using established serological methodology.

Receptor aggregation: A possible mechanism of platelet stimulation by thrombin

The currently accepted view of the structure of biological membranes is of a two dimensional fluid solution of integral proteins embedded in a lipid bilayer (1). Binding of multivalent ligands such as antibodies and lectins often results in rapid lateral mobility followed by clustering of the receptors. Recently, it has been demonstrated that receptor aggregation is important in the action of insulin, a presumably monovalent ligand, on adipocytes (2). Utilizing fluorescent-labeled epidermal growth factor and insulin, aggregation of the receptors on living fibroblasts has also been directly demonstrated (3). It has been shown that the initial step in the interaction of thrombin with platelets is its binding to specific structures on the cell surface (4-9). Considerable data have now accumulated which suggest that the throrabin receptor in human platelets may be a glycoprotein of molecular weight 150,000 (GP-I) (10-12). In our studies on thrombin binding to isolated GP-I, we observed a considerable amount of GP-I aggregates which eluted at the void volume of the column (13). Furthermore, it is known that some lectins can mimick thrombin's ability to stimulate plarelets (14,15). In view of these findings, we considered the possibility that thrombin may lead to clustering of the GP-I molecules on the platelet sur-