Class Of Saturable Binding Sites Research Articles

Candidacidal Activity of Salivary Histatins: IDENTIFICATION OF A HISTATIN 5-BINDING PROTEIN ON Candida albicans

Candida albicans is the predominant species of yeast isolated from patients with oral candidiasis, which is frequently a symptom of human immunodeficiency virus infection and is a criterion for staging and progression of AIDS. Salivary histatins (Hsts) are potent in vitro antifungal agents and have great promise as therapeutic agents in humans with oral candidiasis. The molecular mechanisms by which Hsts kill yeast cells are not known. We report here, that unlike other antimicrobial proteins, Hsts do not display lytic activities to lipid membranes, measured by release and dequenching of the fluorescent dye calcein. Analysis of the magnitude and time course of Hst-induced calcein release from C. albicans cells further showed that loss of cell integrity was a secondary effect following cell death, rather than the result of primary disruption of the yeast cell membrane. 125I-Hst 5 binding studies indicated that C. albicans expressed a class of saturable binding sites (KD = 1 microM), numbering 8.6 x 10(5) sites/cell. Both Hst 3 and Hst 4 competed for these binding sites with similar affinities, which is consistent with the micromolar concentration of Hsts required for candidacidal activity. Specific 125I-Hst 5 binding was not detected to C. albicans spheroplasts, which were 14-fold less susceptible to Hst 5 killing, compared with intact cells in candidacidal assays. In overlay experiments, 125I-Hst 5 bound to a 67-kDa protein detected in C. albicans whole cell lysates and crude membrane fractions, but not in the yeast cell wall fraction. Consistent with the overlay data, cross-linking of 125I-Hst 5 to C. albicans resulted in the appearance of a specific 73-kDa 125I-Hst 5-containing complex that was not detected in the cell wall. 125I-Hst 5-binding protein of similar size was also observed in susceptible S. cerevisiae strain TI#20. This is the first description of Hst 5 binding sites on C. albicans which mediate cell killing and identification of a 67-kDa yeast Hst 5-binding protein. The binding characteristics of Hst 5 are in agreement with the observed potency of its biological effect and provide crucial information to the use of Hst 5 as a therapeutic agent. The presence of a specific C. albicans Hst 5-binding protein provides further insight into the potential mechanism of yeast killing and suggests a basis for differential activity between yeast killing and the nontoxic nature of Hsts to humans.

CCK-JMV-180 acts as an antagonist of the CCK A receptor in the human IMR-32 neuroblastoma cell line

[ 25I]Cholecystokinin-8-S (CCK-8-S) bound to a single class of saturable binding sites on the human neuroblastoma cell line IMR-32 ( K D = 4 ± 1.5 nM, B max = 10,500 ± 3,500 sites/cell ( n = 6)). These binding sites were of the CCK A type, as demonstrated by the differential inhibition of the binding of [ 125I]CCK-8-S and CCK-8-S-induced 45Ca 2+ efflux by the specific CCK A antagonist SR 27897 and the specific CCK B antagonist PD 134,308. CCK-JMV-180, an analogue of CCK-8-S which has been shown to activate 45Ca 2+ efflux in rat cells in a manner similar to CCK-8-S, acted as a potent antagonist of CCK-8-S-induced 45Ca 2+ efflux (IC 50 = 50 nM) and inhibited [ 125I]CCK-8-S binding to IMR-32 cells (IC 50 = 1.7 nM). These results show that, unlike its CCK-like effect in various animal systems, CCK-JMC-180 acts as an antagonist of CCK A receptors in the human neuroblastoma cell line IMR-32.

Platelet LDL receptor recognizes with the same apparent affinity both oxidized and native LDL. Evidence that the receptor-ligand complexes are not internalized.

We have recently demonstrated that the platelet low-density lipoprotein (LDL) receptor is immunologically different from the "classic" receptor of nucleated cells. We undertook the current studies to investigate the interaction of this receptor with oxidized LDL and to determine whether an endocytosis-mediated response is involved in the binding of LDL to platelets. The platelet LDL receptor recognized with the same affinity both native and oxidized LDL particles (IC50, 0.045 and 0.054 g/L; Kd, 45.8 and 65.9 nmol/L, respectively). The Hill coefficients of the displacement of 125I-LDL binding were -1.10 and -1.05 for unlabeled native and oxidized LDL, respectively, thereby suggesting a single set of binding sites. To ascertain whether human platelets bind oxidized LDL, we performed ligand binding assays with 125I-oxidized LDL. Saturation curves of 125I-oxidized LDL binding at 22 degrees C showed that human platelets bound these modified particles to a class of saturable binding sites, numbering approximately 3895 +/- 241 per platelet with a dissociation constant (Kd) of 96.2 +/- 10.3 nmol/L. Displacement experiments showed that 125I-oxidized LDL binding was inhibited with the same affinity by both oxidized and native LDL (IC50, 0.055 and 0.065 g/L; Kd, 88 and 64 nmol/L, respectively). The Hill coefficients of the displacement of the 125I-oxidized LDL binding were -1.02 and -1.07 for unlabeled oxidized and native LDL, respectively, suggesting that a single set of binding sites is implicated. Moreover, oxidized LDL- at a protein concentration of 0.5 g/L enhanced ADP- and collagen-induced platelet aggregation in a manner similar to native LDL.(ABSTRACT TRUNCATED AT 250 WORDS)

Alpha-Fucose-mediated binding and degradation of tissue-type plasminogen activator by HepG2 cells.

The glycoprotein tissue-type plasminogen activator (t-PA) is subject to hepatic clearance in humans. Here, the interaction of t-PA with a well-differentiated hepatoma cell line (HepG2) was examined. Suspended HepG2 cells bound 125I-t-PA in a specific, saturable, and reversible fashion through a Ca(2+)-dependent, active site-independent mechanism. Binding isotherms indicated a high affinity system with a single class of saturable binding sites (Kd 39 nM; maximum binding capacity 493,000 sites per cell). Bound t-PA was rapidly degraded at 37 degrees C in a manner inhibited by lysosomotropic agents or metabolic inhibitors. Pretreatment of t-PA with monoclonal antibodies against the EGF/fibronectin finger domain, but not kringle 2 or kringle 1, reduced total binding by 86%. Binding of 125I-t-PA to HepG2 cells was inhibited by monosaccharides fucose and galactose and by the neoglycoprotein fucosyl-albumin. Enzymatic removal of alpha-fucose residues, but not alpha-galactose, high mannose, or complex oligosaccharide from 125I-t-PA, reduced specific binding by 60 +/- 5%. Binding was also inhibited by high, but not low, molecular weight urokinase, which contains an EGF-based threonine-linked alpha-fucose homologous to that of t-PA. These data suggest that EGF-associated O-linked alpha-fucose may mediate t-PA binding and degradation by HepG2 cells. This mechanism may be relevant to other proteins with analogous structures.

Ritodrine: A β-adrenergic receptor antagonist in human amnion

Human amnion is important in the initiation of labor. Ritodrine, when administered as a tocolytic, is found unchanged in amniotic fluid. We characterized effects of ritodrine binding to beta-adrenergic receptors in amnion and amniocytes. Iodine 125-iodopindolol, beta-adrenergic receptor agonists, and beta-adrenergic receptor antagonists were used to describe binding characteristics. Experiments were designed with and without isoproterenol and ritodrine to study intracellular cyclic adenosine 3'5'-monophosphate and prostaglandin E2 release. Scatchard analysis revealed a single class of saturable binding sites, with maximum binding capacity of 70.0 +/- 17.2 fmol/mg protein (n = 12) and with high-affinity dissociation constant of 458.9 +/- 72.1 pmol/L. Agonists and antagonists competed for the 125I-iodopindolol binding site consistent with a beta 2-adrenergic receptor. Hill coefficients were 0.6 to 0.8 for agonist competition and 1.0 for antagonist competition and ritodrine. Stimulation with isoproterenol resulted in dose-dependent increases in cyclic adenosine 3'5'-monophosphate and prostaglandin E2. Ritodrine failed to stimulate cyclic adenosine 3'5'-monophosphate and inhibited isoproterenol-stimulated cyclic adenosine 3'5'-monophosphate and prostaglandin E2 production. In human amnion binding of ritodrine to beta 2-adrenergic receptors and lack of ritodrine-mediated postreceptor effects are characteristic of a beta 2-adrenergic antagonist.

LDL binding sites on platelets differ from the "classical" receptor of nucleated cells.

Washed human platelets bound radioiodinated low density lipoprotein (125I-LDL) to a class of saturable binding sites; they numbered 1,348 +/- 126 per platelet, and the dissociation constant (KD) was 50.7 +/- 9 nM. 125I-LDL binding to platelets was reversible, and apparent equilibrium was attained within 25 minutes at 22 degrees C and was characterized by forward and reverse rate constants of 1.47 x 10(4) x sec-1 x M-1 and 8 x 10(-4) x sec-1 x M-1, respectively. Such binding was largely unaltered by temperature, divalent ions, and chelating agents. In addition, neither did receptor regulation (up or down) occur when platelets were loaded with cholesterol, nor did prostaglandin E1 (PGE1) increase the binding of 125I-LDL to platelets. On the other hand, the specificity of LDL binding was not typical of the LDL receptor of nucleated cells. Lipoproteins competed for the occupancy of LDL binding sites in platelets with the following order of potency: very low density >> intermediate density > high density subfraction 2. High density lipoprotein subfraction 3, heparin, and PGE1 had no effect on this binding. 125I-LDL binding to lymphocytes and fibroblasts and proteolytic degradation of 125I-LDL by lymphocytes was inhibited by the monoclonal antibody IgG-C7 directed against the LDL receptor to 88%, 85%, and 85% (p < 0.001), respectively. However, with this monoclonal antibody, a blocking effect on neither 125I-LDL binding to platelets nor on LDL-enhanced platelet aggregation induced by ADP and collagen was found. Moreover, we confirmed the existence of LDL binding in platelets from patients with familial hypercholesterolemia. Our results indicate that human platelets bind LDL by saturable sites, which clearly differ from the "classical" LDL receptor in their binding properties, absence of receptor regulation, presence in platelets of familial hypercholesterolemia patients, and the lack of a blocking effect of IgG-C7 on LDL binding and LDL biological activity.

Neuropeptide y receptor in bovine hippocampus is a Y 2 receptor

[ 125I]NPY bound to a single class of saturable binding sites on bovine hippocampus membranes with a K D of 0.1 nM and B max of 165 fmol/mg of protein. The rank order of potency of NPY fragments and other structurally related peptides to inhibit [ 125I]NPY binding was: PYY ≥ NPY ⪢ BPP ≥ APP and NPY > NPY-(13–36) > NPY-(18– 36) ≥ NPY-(20–36) ⪢ NPY-(26–36) > NPY-(free acid). The identity of the NPY binding site was investigated by affinity labeling. Gel electrophoresis followed by autoradiography revealed a band with a mol mass of 50 kDa. Unlabeled NPY or PYY, but not BPP, HPP and APP, inhibited labeling of [ 125I]NPY to the 50 kDa protein band. Moreover, labeling was inhibited by NPY > NPY- (18–36) ≥ NPY-(13–36) ≥ NPY-(20–36) > NPY-(26–36) > NPY- (free acid). The binding of [ 125I]NPY and the intensity of the cross-linked band were reduced in parallel by increasing concentrations of unlabeled NPY (IC 50 = 0.7 nM and 0.6 nM, respectively). These studies demonstrate that bovine hippocampal membranes contain a 50 kDa [ 125I]NPY binding site that has the ligand specificity characteristic of the Y 2 receptor subtype.

Assay and characteristics of the iron binding moiety of reticulocyte endocytic vesicles.

A 59Fe assay was designed to detect an Fe(III) binding capacity in NP-40 solubilized proteins from rabbit reticulocyte endocytic vesicles. The iron binding capacity had an apparent molecular weight as determined by gel exclusion chromatography of 450,000 daltons. The iron binding moiety coincided with the major nontransferrin iron-containing material of endocytic vesicles labeled in vivo by incubation of cells with 59Fe, 125I-labeled transferrin. The material solubilized from vesicles with NP-40 exhibited two classes of saturable binding sites, one with an association constant for 59Fe-citrate of 3.63 x 10(9) M-1 and with 6.6 x 10(-12) moles of iron bound per mg protein and the other with a constant of 3.96 x 10(8) M-1 and 1.0 x 10(-12) moles of iron bound per mg protein. These affinities are sufficient to satisfy the solubility characteristics of Fe(III) at pH 5.0. Most of the 59Fe bound both in vivo and in vitro to the iron binding moiety could be displaced with 56Fe and an equivalent amount of 59Fe could subsequently be rebound in vitro. The iron binding assay was adopted to vesicle proteins separated by SDS-polyacrylamide gel electrophoresis with subsequent transfer to nitrocellulose and revealed an iron binding activity of molecular weight approximately 95,000 daltons.

Binding of cytochalasin B to platelets.

The binding of cytochalasin B (CB) to human platelets and to isolated platelet cytosol and membranes has been analyzed with [3H]CB. High- and low affinity classes of saturable binding sites were associated with intact platelets. Binding at very low concentrations of CB (i.e., high-affinity binding) was partially prevented by 100 mM D-galactose or D-glucose and to a much lesser extent by L-glucose. Binding to platelet cytosol also involved two classes of sites with affinities and capacities similar to those observed with the whole cells. None of this binding, however, was affected by 100 mM D-galactose. Saturable binding to platelet membranes occurred at sites with a uniform binding affinity. Approximately 52% of this binding was prevented by 1 M D-galactose and another 15% by cytochalasin E (CE). We hypothesize that binding in the cytosol is to monomeric (low-affinity) and polymerized (high-affinity) actin, whereas membrane binding (high-affinity only) occurs primarily at sites involved with galactose transport.

Renal tubular vasopressin receptors downregulated by dehydration.

Receptors for arginine vasopressin (AVP) were characterized in tubular epithelial basolateral membranes (BL membranes) prepared from the kidneys of male Sprague-Dawley rats. Association of [3H]AVP was rapid, reversible, and specific. Saturation studies revealed a single class of saturable binding sites with a maximal binding (Bmax) of 184 +/- 15 fmol/mg protein and a KD of 0.61 +/- 0.04 nM. IC50S for AVP, lysine vasopressin, and oxytocin were 0.74 nM, 9.7 nM, and greater than 1 microM, respectively. The V2 receptor antagonist was more than 3,700 times as effective in displacing [3H]AVP than was the V1 antagonist. To investigate the physiological regulation of vasopressin receptors, the effects of elevated levels of circulating AVP on receptor characteristics were studied. Seventy-two-hour water deprivation significantly elevated plasma osmolality and caused an 11.5-fold increase in plasma [AVP]. Scatchard analysis revealed a 38% decrease in the number of AVP receptors on the BL membranes from dehydrated animals. The high-affinity binding sites on the BL membranes fit the pharmacological profile for adenylate cyclase-linked vasopressin receptors (V2), which mediate the antidiuretic action of the hormone. We conclude that physiologically elevated levels of AVP can downregulate vasopressin receptors in the kidney.

High-Affinity Benzodiazepine Binding Sites on Rat Peritoneal Mast Cells and RBL-1 Cells: Binding Characteristics and Effects on Granule Secretion

High-affinity binding sites for benzodiazepines are present in a wide range of tissues other than brain, and appear to be pharmacologically distinct from central nervous system receptors. Binding sites have been reported on rat peritoneal mast cells, but specificity of binding and functional effects have not been addressed in these cells. Using the specific radioligand [3H]-Ro5-4864, which binds to peripheral but not central binding sites, we characterized binding to rat peritoneal mast cells and the related cell line, RBL-1 cells, and effects on granule secretion. Studies in rat peritoneal mast cells disclosed one class of saturable binding sites with a dissociation constant of KD = 5.29 +/- 1.26 nM (mean +/- SE) and maximal binding Bmax = 357 +/- 113 fmol/10(6) cells. Competition studies showed that IC50 for Ro5-4864 was 15.7 nM, for PK11195 was 10.9 nM, for diazepam was 191 nM, and for clonazepam was greater than 10(-5) M. Studies in RBL-1 cells also showed one class of saturable binding sites with KD = 1.11 +/- 0.72 nM and Bmax = 177 +/- 69 fmol/10(6) cells; specificity was similar to peritoneal mast cells. Incubation of peritoneal mast cells with Ro5-4864 (10(-10)-10(-5) M) for 60 min, or with 10(-6) M for 0-60 min, showed no change in histamine or serotonin release either in unstimulated mast cells or those stimulated by compound 48/80 or substance P. Experiments with the fluorescent dye Quin-2 showed no change in free intracellular calcium levels in peritoneal mast cells after incubation with Ro5-4864 ranging from 10(-10) to 10(-6) M.

Identity of Saturable Calcium-Binding Sites on Blood Platelets and Their Involvement in Platelet Aggregation

Extracellular Ca2+ ions are required for platelet aggregation and we show that they enter two platelet pools. One pool is rapidly filled and easily displaced by EGTA. The second is filled more slowly and is not displaced by EGTA. The EGTA-displaceable pool is believed to be surface-located and was found to contain at least one class of saturable binding sites as well as a class of non-saturable binding sites. The saturable sites were found to be highly selective for Ca2+ (dissociation constant, 3.5 X 10(-7) M) even in the presence of 1 mM Mg2+ ions, and they took up between 261,000 and 307,000 Ca2+ ions/platelet. Full occupancy of the saturable binding sites appeared to be necessary for platelet aggregation to proceed. We also studied platelets that were unable to aggregate normally, either due to the congenital bleeding disorder Glanzmann's thrombastenia or due to experimental manipulation. In both cases we found decreased Ca2+ uptake specifically by the saturable Ca2+ binding sites, and that this was associated with decreased number of GP IIb/IIIa molecules expressed on these platelets. We suggest that the Ca2+ binding sites involved in platelet aggregation are located on the GP IIb/IIIa complexes and may be involved in holding the glycoproteins in the complex together, and that the binding sites need to be fully occupied before aggregation can proceed.

Mu-conotoxins share a common binding site with tetrodotoxin/saxitoxin on eel electroplax Na channels

The binding characteristics of conotoxin GIIIA purified from the venom of a marine snail, Conus geographus, with regard to electroplax membranes from Electrophorus electricus were studied using a radiolabeled monopropionyl derivative of the toxin (3H-Pr-CGIIIA). 3H-Pr-CGIIIA bound specifically to a single class of saturable binding sites in electroplax membranes with a dissociation constant of 1.1 +/- 0.2 nM and a maximal binding capacity of 11 +/- 2 pmol/mg of protein. The latter value was similar to the number of specific binding sites (10 +/- 2 pmol/mg of protein) for 3H-lysine-tetrodotoxin (3H-Lys-TTX). Monopropionyl CGIIIA and CGIIIA had similar inhibitory effects on the binding of 3H-Lys-TTX (1 nM) to electroplax membranes with IC50 values of 3.5 and 0.9 nM, respectively. The association and dissociation of 3H-Pr-CGIIIA and electroplax membranes were much slower than those of 3H-Lys-TTX and the membranes. Mu-Conotoxins (CGIIIA and CGIIIB) and guanidinium toxins (TTX and saxitoxin) inhibited 3H-Pr-CGIIIA (1 nM) binding to electroplax membranes with IC50 values of 0.6, 1.1, 7.1, and 2.2 nM, respectively. However, several other kinds of neurotoxins and local anesthetics known to interact with Na channels did not affect 3H-Pr-CGIIIA binding. These findings indicate that mu-conotoxins must be classified in the same group of Na channel inhibitors as guanidinium toxins, since they competed with guanidinium toxins for binding sites on the Na channel. The peptide mu-conotoxins should be useful in studies on the functional and structural domains of Na channel proteins.

Microsomal binding sites for antioestrogens in rat liver. Properties and detergent solubilization.

The properties of an antioestrogen binding site (AEBS), which has high affinity and specificity for nonsteroidal antioestrogens and structurally related compounds, have been studied in rat liver microsomes. When subcellular organelles were separated on Percoll density gradients the distribution of the AEBS paralleled that of NADPH-cytochrome c reductase, indicating that the AEBS is associated with the endoplasmic reticulum. Saturation analysis showed that [3H]tamoxifen was bound to a single class of saturable binding sites in liver microsomes with a KD of 0.9 +/- 0.1 nM at 0 degrees C. The equilibrium KD was not significantly different at 22 degrees C. The KD calculated from the association and dissociation rate constants for [3H]tamoxifen binding at 0 degrees C and 22 degrees C was compatible with the KD measured at equilibrium. Ligand specificity studies using tamoxifen analogues showed qualitatively similar structure-affinity relationships for the AEBS from both rat liver and the MCF 7 breast cancer cell line. In general structural modifications caused correspondingly greater changes in affinity for rat liver AEBS than for MCF 7 AEBS. The AEBS was solubilized from microsomal membranes with sodium cholate. This was the only detergent of nine tested that solubilized the site in high yield without loss of activity. Solubilization using cholate was more effective in the presence of 1 M-NaCl. In the solubilized state there was an apparent loss of [3H]tamoxifen binding activity which could be restored by dilution of the detergent. Gel filtration indicated an Mr of 440,000-490,000 for the AEBS-cholate complex. These studies demonstrate that rat liver contains high concentrations of a microsomal AEBS which has similar properties and specificity to the AEBS previously described in human breast cancer cells. This site can be solubilized by sodium cholate to supply material suitable for further purification.

Muscarinic cholinergic receptors in a rat phaeochromocytoma cell line

The high affinity binding of the muscarinic ligand 3H-N-methylscopolamine ( 3H-NMS) to intact cells of the rat pheochromocytoma cell line PC12 was studied. A single class of saturable binding sites was observed with a density of about 5000 sites per cell, and a pharmacological profile consistent with muscarinic receptors. The inhibition of 3H-NMS binding by the antagonist pirenzepine was of low affinity suggesting PC12 muscarinic receptors may be similar to those found on rat sympathetic nerve terminals.

Changes in surface-bound and exchangeable calcium during platelet activation.

Using 45CaCl2, we measured the effect of ADP and epinephrine on exchangeable Ca2+ pools in gel-filtered platelets. Unstimulated platelets contained 1) a slowly exchangeable pool, representing 54% of the exchangeable Ca2+, that was unaffected by ethylene glycol bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid and was, therefore, presumably intracellular; and 2) a rapidly exchangeable pool, representing 46% of the Ca2+, that appeared to be surface-bound because it was removable by ethylene glycol bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid or LaCl3. Equilibrium studies of this surface-bound Ca2+ revealed two classes of saturable binding sites, one class of 57,000 sites with a high affinity (KD = 5 x 10(-9) M) and another of 460,000 sites with a lower affinity for Ca2+ (KD = 3 x 10(-7) M). Two changes were observed with ADP and epinephrine. First, the amount of surface-bound Ca2+ increased by up to 50%. In the case of ADP, this included an increase in the number of lower affinity Ca2+ binding sites by 28%. Epinephrine increased the number of lower affinity sites by 18% (p less than 0.001). Second, while not increasing the amount of intracellular Ca2+, both agonists increased by 40% the rate of Ca2+ exchange into the intracellular pool. These effects were rapid (less than 1 min), dependent on agonist concentration, and inhibited by prostaglandin I2 or by specific antagonists of ADP or epinephrine binding. Thus, we find no evidence for a net influx of Ca2+ into platelets stimulated by ADP or epinephrine. However, both agonists increase the amount of Ca2+ bound to the platelet surface membrane. This bound Ca2+ may participate in several membrane reactions involved in platelet activation.

Membrane potential dependent binding of scorpion toxin to the action potential sodium ionophore. Studies with a 3-(4-hydroxy 3-[125I] iodophenyl) propionyl derivative.

Abstract— A polypeptide toxin purified 80‐fold from the venom of the scorpion Leiurus quinquestriatus enhances activation of the action potential Na+ ionophore by the alkaloid neurotoxins veratridine, batrachotoxin and aconitine in electrically excitable neuroblastoma cells. The purified toxin can be labelled with [125I] by reaction with N‐succinimidyl 3‐(4‐hydroxy 3‐[125I] iodophenyl) propionate. The [125I] labelled toxin obtained from carboxymethyl Sephadex ion exchange chromatography appears homogeneous by gel electrophoresis and isoelectric focusing. The [125I] labelled toxin binds to a single class of saturable binding sites and also activates the action potential Na+ ionophore in electrically excitable neuroblastoma cells showing identical concentration dependence for both the binding and the activation effects. The labelled toxin does not show any saturable binding or activation of the action potential Na+ ionophore in variant neuroblastoma clones that specifically lack the action potential Na+ ionophore. The results indicate that scorpion toxin binds specifically to the action potential Na+ ionophore. The binding sites have a mean equilibrium dissociation constant of 3 IIH, a mean binding capacity of 46fmol toxin per mg cell protein and a mean density of 24 sites per μm2 of cell surface membrane. A single action potential Na+ ionophore transports 1 × 108 ions per min and has a conductance of 3 psiemens at physiologic ion concentrations. Depolarization of cells by elevated K+ concentration inhibits the saturable binding. Depolarization of cells by incubation in high Na+ medium (130mm‐Na+, 5mm‐K+) with gramicidin A or batrachotoxin also inhibits the saturable toxin binding. These results suggest that scorpion toxin binds specifically to a regulatory component (gate) of the Na+ ionophore. whose conformation is dependent on membrane potential.

Binding to saxitoxin to electrically excitable neuroblastoma cells.

Saxitoxin inhibits the action potential Na+ ionophore of electrically excitable neuroblastoma cells with a KI of 3.7 nM. Binding experiments detect a single class of saturable binding sites with KD = 3.9 nM and a binding capacity of 156 fmol/mg of cell protein (78 sites per micrometer2 of cell surface). Saturable binding is completely inhibited by tetrodotoxin but is unaffected by scorpion toxin or batrachotoxin. No saturable binding is observed in cultures of clone N103, a variant neuroblastoma clone lacking the action potential Na+ response. Thus, saxitoxin binds specifically to the action potential Na+ ionophore in neuroblastoma cells. Comparison of saxitoxin and scorpion toxin binding reveals that there are three saxitoxin receptor sites for each scorpion toxin receptors site. The implications of this stoichiometry are considered.