Loss Of High-affinity Binding Research Articles

Activation-dependent post-transcriptional regulation of CD40L mediates B cell development and survival in germinal centers

Abstract We have previously described a post-transcriptional pathway of induced mRNA stability that occurs at late times of T cell activation and is engaged by the binding of polypyrimidine tract-binding protein (PTBP1) complex to the 3′ UTR of the CD40L transcript. We recently explored the in vivo role of this pathway by generating a genetically modified mouse (CD40LΔ5) bearing a deletion of the PTBP1 stability element. We found that the CD40LΔ5 mice display a dysfunctional germinal center (GC) response including significantly decreased levels of isotype switched antibodies, antibody-secreting cells, memory B cells and GL7+ GC B cells. To extend our earlier findings we measured affinity maturation of antibodies in response to the hapten NP and found a loss of high affinity binding that corresponded to reduced numbers of targeted VH mutations known to be associated with high affinity binding. However, there were no discernible differences in the overall number of mutations across the VH segment, suggesting that the CD40LΔ5 pathway may have a greater effect on the selection and/or expansion of high-affinity B cells rather than on the somatic hypermutation (SHM) in general. To identify how the CD40L mRNA stability pathway impacted B cells during an immune response, CD19+ B cells from immunized mice were transcriptionally profiled using RNAseq. Expression of genes associated with cell survival and proliferation were found to be down-regulated and apoptotic genes up-regulated in CD40LΔ5 B cells compared to WT cells. Together these finding are consistent with the Δ5 stability element playing a critical role in the proliferation and cell survival of high affinity GC B cells through a pathway of enhanced expression of CD40L in CD4 T cells.

Replacement of the bryostatin A- and B-pyran rings with phenyl rings leads to loss of high affinity binding with PKC

We describe a convergent synthesis of a bryostatin analogue in which the natural A- and B-ring pyrans have been replaced by phenyl rings. The new analogue exhibited PMA like behavior in cell assays, but failed to maintain high affinity binding for PKC, despite retaining an unaltered C-ring ‘binding domain’

THU0058 B Cell Receptor Editing in Scleroderma Patients Generates Pathogenic Anti-PDGFR Autoantibodies

BackgroundSystemic sclerosis or scleroderma (SSc) is a clinically heterogeneous disease of the connective tissue characterized by vascular, immune/inflammatory and fibrotic manifestations. We have hypothesized that the serum of SSc patients...

The Role of Interchain Heterodisulfide Formation in Activation of the Human Common β and Mouse βIL-3 Receptors

The cytokines, interleukin-3 (IL-3), interleukin-5 (IL-5), and granulocyte-macrophage colony-stimulating factor (GM-CSF), exhibit overlapping activities in the regulation of hematopoietic cells. In humans, the common beta (betac) receptor is shared by the three cytokines and functions together with cytokine-specific alpha subunits in signaling. A widely accepted hypothesis is that receptor activation requires heterodisulfide formation between the domain 1 D-E loop disulfide in human betac (hbetac) and unidentified cysteine residues in the N-terminal domains of the alpha receptors. Since the development of this hypothesis, new data have been obtained showing that domain 1 of hbetac is part of the cytokine binding epitope of this receptor and that an IL-3Ralpha isoform lacking the N-terminal Ig-like domain (the "SP2" isoform) is competent for signaling. We therefore investigated whether distortion of the domain 1-domain 4 ligand-binding epitope in hbetac and the related mouse receptor, beta(IL-3), could account for the loss of receptor signaling when the domain 1 D-E loop disulfide is disrupted. Indeed, mutation of the disulfide in hbetac led to both a complete loss of high affinity binding with the human IL-3Ralpha SP2 isoform and of downstream signaling. Mutation of the orthologous residues in the mouse IL-3-specific receptor, beta(IL-3), not only precluded direct binding of mouse IL-3 but also resulted in complete loss of high affinity binding and signaling with the mouse IL-3Ralpha SP2 isoform. Our data are most consistent with a role for the domain 1 D-E loop disulfide of hbetac and beta(IL-3) in maintaining the precise positions of ligand-binding residues necessary for normal high affinity binding and signaling.

Structural Changes in the Lectin Domain of CD23, the Low-Affinity IgE Receptor, upon Calcium Binding

CD23, the low-affinity receptor for IgE (Fc epsilonRII), regulates IgE synthesis and also mediates IgE-dependent antigen transport and processing. CD23 is a unique Fc receptor belonging to the C-type lectin-like domain superfamily and binds IgE in an unusual, non-lectin-like manner, requiring calcium but not carbohydrate. We have solved the high-resolution crystal structures of the human CD23 lectin domain in the presence and absence of Ca2+. The crystal structures differ significantly from a previously determined NMR structure and show that calcium binding occurs at the principal binding site, but not at an auxiliary site that appears to be absent in human CD23. Conformational differences between the apo and Ca2+ bound structures suggest how IgE-Fc binding can be both calcium-dependent and carbohydrate-independent.

Inhibition of Glucocorticoid Receptor Nucleocytoplasmic Shuttling by Okadaic Acid Requires Intact Cytoskeleton

It has been shown previously that glucocorticoid receptors (GRs) that have undergone hormone-dependent translocation to the nucleus and have subsequently exited the nucleus upon hormone withdrawal are unable to recycle into the nucleus if cells are treated during hormone withdrawal with okadaic acid, a cell-permeable inhibitor of certain serine/threonine protein phosphatases. Using a green fluorescent protein (GFP) GR chimera (GFP-GR), we report here that okadaic acid inhibition of steroid-dependent receptor recycling to the nucleus is abrogated in cells treated for 1 h with colcemid to eliminate microtubule networks prior to steroid addition. After withdrawal of colcemid, normal cytoskeletal architecture is restored and okadaic acid inhibition of steroid-dependent GFP-GR nuclear recycling is restored. When okadaic acid is present during hormone withdrawal, GR that is recycled to the cytoplasm becomes complexed with hsp90 and binds steroid, but it does not undergo the normal agonist-dependent dissociation from hsp90 upon retreatment with steroid. However, when the cytoskeleton is disrupted by colcemid, the GR in okadaic acid-treated cells recycles from the cytoplasm to the nucleus in an agonist-dependent manner without dissociating from hsp90. This suggests that under physiological conditions where the cytoskeleton is intact, a dephosphorylation event is required for loss of high affinity binding to hsp90 that is required for receptor translocation through the cytoplasm to the nucleus along cytoskeletal tracts.

Leukotriene receptors

The current challenge in research on leukotriene receptors is to clone these molecules. Traditional protein purification approaches have not been successful in providing sequence information. Solubilization of cys-LT1 has been achieved but results in the dissociation of G-proteins and the loss of high affinity binding (Mong et al., 1986b; Mong and Sarau, 1990), while cys-LT2 activity cannot be monitored by other than functional assays and there have not been any purification attempts. Partial purification of B-LT has been reported but has not been continued to homogeneity (Sherman et al., 1992; Votta et al., 1990; Miki et al., 1990). Nor have attempts to clone these receptors through either homology screening or expression cloning been successful. The cloning of the prostanoid receptors, described in detail elsewhere in this volume, has shown that these receptors belong to a distinct family within the G-protein-coupled receptor superfamily. It is probable, therefore, that the leukotriene receptors will also belong to a separate group within this superfamily since phylogenic comparisons have shown that receptors displaying high affinity for structurally related ligands exist as discrete families. Recently, a human cDNA encoding an orphan FMLP-related receptor cloned from HL60 cells of myeloid lineage was identified as the receptor for another eicosanoid, lipoxin A (Fiore et al., 1994). FMLP has a similar profile of biological actions to LTB4. Moreover, LTD4 showed a high degree of cross-reactivity with this receptor with an affinity only 20-fold less that of lipoxin A, although LTB4 was inactive. It remains to be determined whether the leukotriene receptors will fall into this class of receptors. The cloning of the leukotriene receptors will allow identification of the different receptor types and subtypes and potentially splice variants. Evaluation of currently developed antagonists at these receptor types could also open the way for novel therapies for inflammatory conditions.

An Acidic Motif within the Third Intracellular Loop of the .alpha.2C2 Adrenergic Receptor Is Required for Agonist-Promoted Phosphorylation and Desensitization

The alpha 2C2 adrenergic receptor contains a highly acidic stretch of amino acids (EDEAEEEEEEEEEEEE) within the third intracellular loop. To investigate the role of this region, we utilized site-directed mutagenesis to delete these 16 amino acids as well as to substitute them with glutamine, thereby conserving size but not charge. The wild-type and mutated alpha 2C2 receptors were permanently expressed in CHO cells. Neither substitution nor deletion of this region affected receptor expression, agonist or antagonist binding affinities, guanine nucleotide-sensitive formation of the high-affinity agonist-receptor-G protein complex, or functional coupling of the receptor to Gi. We considered that since alpha 2C2 agonist-promoted desensitization is due to phosphorylation by the beta-adrenergic receptor kinase (or a related kinase), that this region may be important for establishing the acidic mileau required by this kinase. Therefore, the consequences of 30 min of agonist preexposure on subsequent alpha 2C2-mediated inhibition of adenylyl cyclase and on high-affinity agonist binding were determined for the wild-type and these two mutants. The wild-type alpha 2C2 receptor underwent approximately 52% functional desensitization and a approximately 40% loss of high-affinity binding after such exposure. In contrast, deletion and substitution of this acidic stretch of amino acids ablated desensitization as assessed by both approaches. These results correlated with those obtained in whole cell phosphorylation experiments. Cells expressing each receptor were incubated with [32P]orthophosphate and exposed to agonist, and receptors were purified by immunoprecipitation. The deletion and the substitution mutant receptors underwent agonist-promoted phosphorylation at levels of only 44 +/- 5% and 50 +/- 15%, respectively, relative to wild-type alpha 2C2.(ABSTRACT TRUNCATED AT 250 WORDS)

Neutrophils negatively regulate neutropoiesis by increasing the degradation of G-CSF through endocytosis as well as by decreasing its production from blood mononuclear cells

The low- and high-affinity binding of tritiated benzimidazole anthelmintics (mebendazole and oxibendazole) to tubulin-containing supernatants derived from unembryonated eggs, third stage larvae or adult worms of thiabendazole-susceptible and -resistant strains of Haemonchus contortus were examined and compared. The displacement of these radioligands by unlabelled benzimidazoles (mebendazole, fenbendazole, thiabendazole and oxibendazole) also was examined. The binding affinity, Ka, and maximum binding, Bmax, for the high-affinity binding were calculated by non-linear least-square iterative curve fitting using a computer programme (LIGAND) based on the exact mathematical model of ligand-receptor interactions. The Ka was of the same order of magnitude (×107M−1) for the susceptible and resistant eggs, larvae and worms. Resistance was associated with a loss of high-affinity binding. There was a 2- to 5-fold loss of Bmax by the resistant strain. The eggs showed greater high-affinity binding per milligram of protein than the larvae which, in turn, showed greater high-affinity binding than the adult worms. It was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis that the tubulin content per milligram of protein decreased from egg, through larva to adult worm. Cross-displacement studies indicated that different benzimidazole drugs interacted with the same receptor (tubulin) and that a rank order of affinity of the benzimidazole drugs could be inferred.

Identification of basic residues involved in activation and calmodulin binding of rabbit smooth muscle myosin light chain kinase.

It is postulated that basic residues in the regulatory region of myosin light chain kinase are important for conferring autoinhibition by binding to the catalytic core. To investigate this proposal, 10 basic amino acids within the regulatory region of rabbit smooth muscle myosin light chain kinase (Lys961-Lys979) were replaced either singularly or in combination with acidic or nonpolar residues by site-directed mutagenesis. All active mutant kinases were dependent on Ca2+/calmodulin for catalytic activity. None of the mutants was active in the absence of Ca2+/calmodulin, suggesting that the autoinhibitory region has not been defined completely. Charge reversal mutants at Arg974, Arg975, and Lys976 resulted in loss of high affinity binding of calmodulin and increased the concentration of calmodulin required for half-maximal activation (KCaM). The charge reversal mutant at Lys979 also increased KCaM but to a lesser extent. Charge reversal mutants at Lys965 and Arg967 resulted in an inactive myosin light chain kinase that could not be proteolytically activated. When these residues were mutated to Ala, the expressed kinase was dependent upon Ca2+/calmodulin for activity and exhibited a decrease in KCaM. Charge reversal mutants in Lys961 and Lys962 also had decreased KCaM values. These basic residues amino-terminal of the calmodulin binding domain may play an important role in the activation of the kinase.

The interaction of a kainate receptor from goldfish brain with a pertussis toxin-sensitive GTP binding protein.

Kainate receptors are present in high concentrations in goldfish brain (Henley and Oswald, 1988a and b; Ziegra et al., 1990), possibly in neuronal and glial cells. In a number of systems, the kainate receptor has been assumed to be an integral ion channel (Watkins and Evans, 1981); but, for some kainate receptors, ion channel activity has not been demonstrated (Wada et al., 1989). This study presents evidence that a portion of the [3H]kainate-binding sites in goldfish brain is sensitive to guanine nucleotides, with a loss of high affinity binding in the presence of nonhydrolyzable GTP analogs. Pertussis toxin pretreatment of membranes causes a loss of high affinity [3H]kainate binding and of the guanine nucleotide-sensitive binding. Pertussis toxin catalyzes the specific [32P]ADP-ribosylation of a 40-kDa substrate in a kainate-sensitive manner. In addition, incorporation of [alpha-32P]GTP-gamma-azidoanilide by photoaffinity labeling was enhanced in the presence of kainate. These results indicate that a subpopulation of [3H]kainate-binding sites in goldfish brain may be coupled to G proteins.

The interaction of a kainate receptor from goldfish brain with a pertussis toxin-sensitive GTP-binding protein

Kainate receptors are present in high concentrations in goldfish brain (Henley and Oswald, 1988a and b; Ziegra et al., 1990), possibly in neuronal and glial cells. In a number of systems, the kainate receptor has been assumed to be an integral ion channel (Watkins and Evans, 1981); but, for some kainate receptors, ion channel activity has not been demonstrated (Wada et al., 1989). This study presents evidence that a portion of the [3H]kainate-binding sites in goldfish brain is sensitive to guanine nucleotides, with a loss of high affinity binding in the presence of nonhydrolyzable GTP analogs. Pertussis toxin pretreatment of membranes causes a loss of high affinity [3H]kainate binding and of the guanine nucleotide-sensitive binding. Pertussis toxin catalyzes the specific [32P]ADP-ribosylation of a 40-kDa substrate in a kainate-sensitive manner. In addition, incorporation of [alpha-32P]GTP-gamma-azidoanilide by photoaffinity labeling was enhanced in the presence of kainate. These results indicate that a subpopulation of [3H]kainate-binding sites in goldfish brain may be coupled to G proteins.

Specific interaction of benzimidazole anthelmintics with tubulin from developing stages of thiabendazole-susceptible and -resistant Haemonchus contortus

The low- and high-affinity binding of tritiated benzimidazole anthelmintics (mebendazole and oxibendazole) to tubulin-containing supernatants derived from unembryonated eggs, third stage larvae or adult worms of thiabendazole-susceptible and -resistant strains of Haemonchus contortus were examined and compared. The displacement of these radioligands by unlabelled benzimidazoles (mebendazole, fenbendazole, thiabendazole and oxibendazole) also was examined. The binding affinity, K a , and maximum binding, B max, for the high-affinity binding were calculated by non-linear least-square iterative curve fitting using a computer programme (LIGAND) based on the exact mathematical model of ligand-receptor interactions. The K a was of the same order of magnitude (×10 7M −1) for the susceptible and resistant eggs, larvae and worms. Resistance was associated with a loss of high-affinity binding. There was a 2- to 5-fold loss of B max by the resistant strain. The eggs showed greater high-affinity binding per milligram of protein than the larvae which, in turn, showed greater high-affinity binding than the adult worms. It was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis that the tubulin content per milligram of protein decreased from egg, through larva to adult worm. Cross-displacement studies indicated that different benzimidazole drugs interacted with the same receptor (tubulin) and that a rank order of affinity of the benzimidazole drugs could be inferred.

Deletion of cytoplasmic sequences of the nerve growth factor receptor leads to loss of high affinity ligand binding.

The nerve growth factor (NGF) receptor is a glycosylated transmembrane protein present on the cell surface as both high and low affinity forms, but biological responsiveness requires interactions of NGF with the high affinity site. We have tested the effects of mutations in the intracellular domain of the receptor upon its cell surface expression and equilibrium binding of 125I-NGF. Although mutant receptors lacking the entire cytoplasmic domain are processed and expressed at the cell surface and are capable of binding to NGF, the absence of cytoplasmic sequences leads to a loss of high affinity binding and to a lack of an appropriate cross-linking pattern as assessed by N-hydroxysuccinimidyl 4-azidobenzoate photoaffinity cross-linking. These results, taken together with the highly conserved nature of these cytoplasmic sequences, implies that the interaction of the receptor with an accessory molecule is necessary to form the high affinity receptor.

Coupling of the α1-adrenergic receptor to a guanine nucleotide-binding regulatory protein by a discrete domain distinct from its ligand recognition site

At rat hepatic membrane α 1-adrenergic receptors, the nonhydrolyzable GTP analogue p[NH]ppG causes a rightward shift of agonist competition curves and a loss of high-affinity binding. This p[NH]ppG effect is consistent with the involvement of a guanine nucleotide-binding regulatory protein (G-protein) in α 1-adrenergic receptor signalling. Although readily apparent in membranes prepared to avoid retention of endogenous nucleotides and activation of Ca 2+-sensitive proteinases (+pi), this p[NH]ppG effect is not observed in membranes prepared without proteinase inhibitors (−pi), or in −pi membranes treated with Ca 2+ (−pi, +Ca 2+). In these various membrane preparations, different M r forms of the receptor are also identified by photoaffinity labelling with [ 125I]CP65 526, an aryl azide analog of the α 1-selective antagonist, prazosin, followed by SDS-polyacrylamide gel electrophoresis and autoradiography. Whereas a predominant M r = 80 000 subunit is identified in +pi membranes, in −pi membranes a proteolytic M r = 59 000 fragment is also observed. In −pi, +Ca 2+ membranes, only this latter peptide is detected. To evaluate the ability of each of these forms of the receptor to couple with a G-protein, the effect of p[NH]ppG on the agonist-inhibition of [ 125I]CP65 526 labelling was determined by laser densitometry scanning and computer analysis. At the M r = 80 000 subunit, p[NH]ppG causes a rightward shift of agonist competition curves and a loss of high-affinity binding, even in −pi membranes. By contrast, agonist-binding at the M r = 59 000 subunit is of low-affinity and was not affected by p[NH]ppG. These data indicate that the cleaved M r = 59 000 fragment, while retaining hormone binding activity is unable to undergo G-protein coupling. Thus, the α 1-adrenergic receptor appears to contain a discrete domain necessary for G-protein coupling that is distinct from its ligand recognition site.

Characterization of vasoactive intestinal peptide receptors by a photoaffinity label. Site-specific modification of vasoactive intestinal peptide by derivatization of the receptor-bound peptide.

The biological effects of vasoactive intestinal peptide (VIP) are mediated by binding to a membrane-bound receptor. Probes designed to trap this receptor by binding to it in a covalent way may suffer from a greatly reduced affinity. We report here, for the VIP receptor, the use of a photoaffinity probe obtained by derivatization of receptor-bound VIP with para-azidophenylglyoxal. This method protected the parts of the molecule essential for receptor binding. The VIP derivative thus obtained became covalently linked when irradiated. In the dark, however, it exhibited normal VIP-like behavior and retained its biological activity. This derivatization method might be generally applicable when hormone analogues have to be prepared without loss of receptor affinity. Receptor characterization studies on liver plasma membranes showed the presence of high- and low-affinity binding sites with KD = 0.1 and 5 nM, respectively. Treatment of membranes with dithiothreitol causes loss of high-affinity binding. The high-affinity site, trapped by the photoaffinity probe, resolved into two molecular mass forms, 50 and 200-250 kDa. Reduction of the receptor-probe complex left the 50-kDa form intact, whereas the amount of the 200-250-kDa form greatly diminished. We demonstrate the importance of the presence of disulfide bonds in one of the molecular forms involved in high-affinity binding.

Agonist interactions at hepatic .alpha.1- and .beta.-adrenergic receptors: affinity-state regulation by guanine nucleotides and temperature

We investigated the binding characteristics of agonists to alpha 1- and beta-adrenergic receptors of intact liver cells, broken rat liver cell membranes, and detergent-solubilized preparations under varying experimental conditions, focusing on the different "states" of the receptor for agonists and the regulation of these states by temperature and guanine nucleotides. While only low-affinity binding of agonists to both receptor subtypes was evident in studies performed at 37 degrees C with solubilized preparations, biphasic competition curves for agonists were observed in both intact cells and membrane preparations; the majority of sites were of low affinity. In membrane preparations, the nonhydrolyzable GTP analogue Gpp(NH)p caused a rightward shift of agonist competition curves and a loss of high-affinity binding. These results are consistent with the involvement of guanine nucleotide binding proteins in both alpha 1- and beta-adrenergic transduction pathways. When competition studies were performed at 4 degrees C, receptor sites existed predominantly in the high-affinity configuration, in intact cells and membranes, as well as in soluble preparations. In contrast to the studies conducted at 37 degrees C, no Gpp(NH)p-induced conversion to the lower affinity state could be demonstrated in studies performed with membrane preparations at 4 degrees C. Thus, the high-affinity state of alpha 1- and beta-adrenergic receptors is stabilized at 4 degrees C in intact cells, membranes, and soluble preparations. After incubations had been performed at 37 degrees C, high-affinity binding of agonists could not be restored by subsequent incubation at 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Somatogenic receptors of rat liver: regulation by insulin.

Somatogenic (i.e. GH) receptors have been studied on liver microsomal membranes from male and female rats. Tracer bovine GH was displaced from its binding sites by GHs of various species, but was displaced only weakly by PRLs. Specific bovine GH binding was 3.5-fold higher to female rat liver membranes than to membranes from males. Streptozotocin-induced diabetes significantly reduced binding, by 80% in females and 50% in males, while insulin therapy to normalize weight gain reversed the decrease in binding. Competitive binding curves were consistent with two independent classes of binding site: low affinity sites with K equal to 0.5 nm-1 in both sexes, and high affinity sites with K equal to 12.1 nm-1 in males and 21.4 nm-1 in females (P less than 0.001). The addition of excess ovine PRL caused a substantial loss of high affinity binding with little loss in the low affinity region, suggesting a weak somatogenic role for ovine PRL. In diabetic animals, low affinity sites were unchanged from normal, while high affinity sites were decreased in number, with no change in affinity, and restored on insulin therapy. Serum immunoreactive rat GH levels were the same in normal and diabetic, male and female animals. These studies suggest that the apparent hepatic resistance to GH seen in diabetes when liver somatomedin release is low despite normal serum GH might be explained by the loss of GH receptors in this condition.

Binding of scorpion toxin to sodium channels in vitro and its modification by beta-bungarotoxin.

1. Binding of a purified scorpion toxin to membrane fragments isolated from electroplaque of an electric eel Electrophorus electricus was studied using a radio-iodinated toxin.2. A scorpion toxin was purified from the venom of Leiurus quinquestriatus and iodinated with (125)I in a lactoperoxidase-catalysed reaction. Monoiodinated toxin, isolated by an ion exchange chromatography, retarded the inactivation kinetics of Na current to a similar extent as the native toxin, indicating that radioiodination did not appreciably affect physiological and binding properties of the native toxin.3. Analyses of binding properties by Scatchard plots showed the presence of two classes of binding sites (with low and high affinities) in the membrane preparation from eel electroplaque; similar preparation from an electric skate, of which the electroplaque is known to be devoid of Na channels, possessed only the low affinity sites.4. The number of high affinity sites in the eel preparation was 41.8 +/- 10.5 p-mole/g tissue; the value was within the range reported for tetrodotoxin binding to similar preparations (15-148 p-mole/g tissue).5. A variety of cations (Na(+), Mn(2+) and La(3+)) inhibited the high affinity scorpion toxin binding, as indicated for the toxin binding to Na channels by a previous electrophysiological study. K(D) value in the presence of 120 mM-Na(+) (approx. 8 nM) agreed reasonably with that (approx. 10nM) reported for the scorpion toxin binding to excitable neuroblastoma cells or synaptic nerve ending particles under conditions where membrane potential was depolarized by the addition of 135 mM-KCl.6. Pretreatment of the eel membrane preparation with beta-bungarotoxin (7-44 ng/ml.) in the presence of Ca ions (10-200 muM) resulted in a substantial loss of high affinity binding of scorpion toxin. When phospholipase A(2) activity of the beta-toxin was inactivated by a chemical modification with p-bromophenacyl bromide, the inhibitory action of the beta-bungarotoxin was abolished.7. It is concluded that a high affinity binding of scorpion toxin to the eel electroplaque membrane fragments represents the binding to Na channels in vitro, and that phospholipase A(2) activity of beta-bungarotoxin interferes with the binding of scorpion toxin to Na channels.

Structure-function relationships in the interaction of alpha-thrombin with blood platelets.

Highly purified alpha-thrombin has been chemically modified in an attempt to determine which features of the molecule are important for normal platelet-thrombin interactions. Modifying agents included diisopropylphosphorofluoridate and 1-chloro-3-tosylamido-7-amino-L-2-heptanone, which modify serine and histidine, respectively, at the catalytic site, as well as N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide, which modify a single tryptophan at or near the fibrinogen-binding site. Active site-directed modification did not appreciably affect the binding characteristics, but prevented platelet activation. In contrast, modification of tryptophan at the macromolecular substrate-binding site resulted in the loss of high affinity binding of thrombin to platelets, while low affinity binding was apparently unaffected. This modification altered but did not abolish the ability of thrombin to effect platelet aggregation and release of [14C]serotonin. These results suggest that residues at the catalytic site are not involved in binding and that the macromolecular substrate-binding site of alpha-thrombin participates in high affinity binding to platelets. These data are also consistent with the existence of at least two types of binding sites for thrombin on the platelet surface as well as more than one platelet-binding region on the thrombin molecule.