Time Course Of Association Research Articles

The Kinetics of Cooperative Cofilin Binding Reveals Two States of the Cofilin-Actin Filament

The interaction of cofilin with actin filaments displays positive cooperativity. The equilibrium binding and associated thermodynamic properties of this interaction are well described by a simple, one-dimensional Ising model with nearest neighbor interactions. Here we evaluate the kinetic contributions to cooperative binding and the ability of this model to account for binding across a wide range of cofilin concentrations. A Monte Carlo-based simulation protocol that allows for nearest-neighbor interactions between adjacent binding sites was used to globally fit time courses of human cofilin binding to human nonmuscle (β-, γ-) actin filaments. Several extensions of the one-dimensional Ising model were tested, and a mechanism that includes isomerization of the actin filament was found to best account for time courses of association as well as irreversible dissociation from a saturated filament. This model predicts two equilibrium states of the cofilin-actin, or cofilactin, filament, and the resulting set of binding parameters are in agreement with equilibrium thermodynamic parameters. We conclude that despite its simplicity, this one-dimensional Ising model is a reliable model for analyzing and interpreting the energetics and kinetics of cooperative cofilin-actin filament interactions. The model predicts that severing activity associated with boundaries between bare and decorated segments will not be linear, but display a transient burst at short times on cofilin activation then dissipate due to a kinetic competition between severing activity and cofilin binding. A second peak of severing activity is predicted to arise from irreversible cofilin dissociation on inactivation. These behaviors predict what we believe to be novel mechanisms of cofilin severing and spatial regulation of actin filament turnover in cells. The methods developed for this system are generally applicable to the kinetic analysis of cooperative ligand binding to linear polymers.

Kinetics of the Interaction of myo1c with Phosphoinositides

myo1c is a single-headed myosin that dynamically links membranes to the actin cytoskeleton. A putative pleckstrin homology domain has been identified in the myo1c tail that binds phosphoinositides and soluble inositol phosphates with high affinity. However, the kinetics of association and dissociation and the influence of phospholipid composition on the kinetics have not been determined. Stopped-flow spectroscopy was used to measure the binding and dissociation of a recombinant myo1c construct containing the tail and regulatory domains (myo1c(IQ-tail)) to and from 100-nm diameter large unilamellar vesicles (LUVs). We found the time course of association of myo1c(IQ-tail) with LUVs containing 2% phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) followed a two-exponential time course, and the rate of the predominant fast phase depended linearly upon the total lipid concentration. The apparent second-order rate constant was approximately diffusion-limited. Increasing the molar ratio of anionic phospholipid by adding phosphatidylserine, additional PtdIns(4,5)P(2), or by situating PtdIns(4,5)P(2) in a more physiologically relevant lipid background increased the apparent association rate constant less than 2-fold. myo1c(IQ-tail) dissociated from PtdIns(4,5)P(2) at a slower rate (2.0 s(-1)) than the pleckstrin homology domain of phospholipase C-delta (13 s(-1)). The presence of additional anionic phospholipid reduced the myo1c(IQ-tail) dissociation rate constant >50-fold but marginally changed the dissociation rate of phospholipase C-delta, suggesting that additional electrostatic interactions in myo1c(IQ-tail) help to stabilize binding. Remarkably, high concentrations of soluble inositol phosphates induce dissociation of myo1c(IQ-tail) from LUVs, suggesting that phosphoinositides are able to bind to and dissociate from myo1c(IQ-tail) as it remains bound to the membrane.

Loneliness and cortisol: Momentary, day-to-day, and trait associations

In attempts to understand the social determinants of health, strong associations have been found between measures of loneliness, physiological stress processes, and physical and mental health outcomes. Feelings of loneliness are hypothesized to have implications for physiological stress processes, including activity of the hypothalamic-pituitary-adrenal (HPA) axis. In a community sample of young adults, multilevel modeling was used to examine whether trait and state feelings of loneliness were related to changes in levels of the stress-sensitive hormone cortisol, and whether the associations between loneliness and cortisol were mediated or moderated by the presence of concurrent depression or high levels of chronic life stress. Results indicated that trait loneliness was associated with a flattening of the diurnal cortisol rhythm. In addition, both daily and momentary state variations in loneliness were related to cortisol. Prior day feelings of loneliness were associated with an increased cortisol awakening response the next morning and momentary experiences of loneliness during the day were associated with momentary increases in cortisol among youth who also had high chronic interpersonal stress. Results were significant after covarying current depression, both chronic and momentary reports of stress, and medical and lifestyle covariates. This study expanded on prior work by investigating and revealing three different time courses of association between loneliness and HPA axis activity in young adults: trait, daily and momentary.

Widely Distributed Residues in Thymosin β4 Are Critical for Actin Binding

We have investigated the contributions of hydrophobic residues, the conserved and variable proline residues, and the conserved lysine residues to the affinity and kinetics of thymosin beta4 (Tbeta4) binding to MgATP-actin monomers. Pro4, Lys18, Lys19, Pro27, Leu28, Pro29, and Ile34 were substituted with alanine residues. Mutagenesis of Pro4 or Pro27 has little effect (<or=3-fold reduction) on the actin binding affinity of Tbeta4. Substitution of Lys18 and Lys19, Leu28, Pro29, or Ile34 weakens the affinity of the actin-Tbeta4 complex >or=10-fold, but the kinetic basis of the lower stability varies among the mutants. Substitution of the conserved lysine residues weakens the affinity by slowing association and accelerating dissociation. Substitution of hydrophobic residue Leu28 or Ile34 weakens the affinity by accelerating dissociation. These results favor a reaction mechanism in which Tbeta4 binds actin monomers following a two-step mechanism in which the formation of a bimolecular complex is followed by isomerization to a strong binding state that is coupled to the formation of widely distributed hydrophobic contacts. The isomerization equilibrium is slowed by mutagenesis of Pro29, as revealed by the double-exponential time course of association. Mutagenesis of Pro4 or Pro27 accelerates binding and dissociation but minimally affects the binding affinity (<or=3-fold reduction), suggesting that cis- trans isomerization of these proline residues contributes to the slow association rate constant of wild-type Tbeta4.

Mechanisms of Pharmacological Rescue of Trafficking-defective hERG Mutant Channels in Human Long QT Syndrome

Long QT syndrome type 2 is caused by mutations in the human ether-a-go-go-related gene (hERG). We previously reported that the N470D mutation is retained in the endoplasmic reticulum (ER) but can be rescued to the plasma membrane by hERG channel blocker E-4031. The mechanisms of ER retention and how E-4031 rescues the N470D mutant are poorly understood. In this study, we investigated the interaction of hERG channels with the ER chaperone protein calnexin. Using coimmunoprecipitation, we showed that the immature forms of both wild type hERG and N470D associated with calnexin. The association required N-linked glycosylation of hERG channels. Pulse-chase analysis revealed that N470D had a prolonged association with calnexin compared with wild type hERG and E-4031 shortened the time course of calnexin association with N470D. To test whether the prolonged association of N470D with calnexin is due to defective folding of mutant channels, we studied hERG channel folding using the trypsin digestion method. We found that N470D and the immature form of wild type hERG were more sensitive to trypsin digestion than the mature form of wild type hERG. In the presence of E-4031, N470D became more resistant to trypsin even when its ER-to-Golgi transport was blocked by brefeldin A. These results suggest that defective folding of N470D contributes to its prolonged association with calnexin and ER retention and that E-4031 may restore proper folding of the N470D channel leading to its cell surface expression.

Calmodulin Dissociation Mediates Desensitization of the cADPR-Induced Ca2+ Release Mechanism

Ryanodine receptor (RyR) activation by cyclic ADP-ribose (cADPR) is followed by homologous desensitization [1–3]. Though poorly understood, this “switching off” process has provided a key experimental tool for determining the pathway through which cADPR mediates Ca2+ release [4]. Moreover, desensitization is likely to play an important role in shaping the complexities of Ca2+ signaling involving cADPR, for example, localized release events [5, 6] and propagated waves [7–9]. Using the sea urchin egg, we unmask a role of calmodulin, a component of the RyR complex [10, 11] and a key cofactor for cADPR activity [12–14], during RyR/cADPR desensitization. Recovery from desensitization in calmodulin-depleted purified endoplasmic reticulum (microsomes) is severely impaired compared to that in crude egg homogenates. An active, soluble factor, identified as calmodulin, is required to restore the capacity of microsomes to recover from desensitization. Calmodulin mediates recovery in a manner that tightly parallels its time course of association with the RyR. Conversely, direct measurement of calmodulin binding to microsomes reveals a loss of specific binding during cADPR, but not IP3, desensitization. Our results support a mechanism in which cycles of calmodulin dissociation and reassociation to an endoplasmic reticulum protein, most likely the RyR itself, mediate RyR/cADPR desensitization and resensitization, respectively.

Kinetic Analysis of the Interaction between HIV-1 Protease and Inhibitors Using Optical Biosensor Technology

The interaction between HIV-1 protease and reversible inhibitors was studied by surface plasmon resonance biosensor technology. The steady-state binding level and the time course of association and dissociation could be observed by measuring the binding of inhibitors injected in a continuous flow of buffer to the immobilized enzyme. Fourteen low molecular weight inhibitors (500–700 Da), including the four clinically used HIV-1 protease inhibitors (indinavir, nelfinavir, ritonavir, and saquinavir), were analyzed. Affinities were estimated as B50 values from a series of sensorgrams at different concentrations of inhibitors. These values were found to be correlated with inhibition constants (Ki) determined by an enzyme inhibition assay (r2 = 0.84, logarithmic values). Dissociation rates were estimated at a single saturating concentration of the inhibitors as t1/2,obs, but these values did not correlate with Ki (r2 = 0.26, logarithmic values). Indinavir had the highest affinity (B50 = 11 nM) and the fastest dissociation (t1/2,obs = 500 s) among the clinically used inhibitors while saquinavir had a lower affinity (B50 = 25 nM) and the slowest dissociation rate (t1/2,obs = 6500 s). Since these two inhibitors have similar Ki values, the differences in dissociation rates reveal important characteristics in the interaction that cannot be obtained by the inhibition studies. The biosensor data are expected to be of greater in vivo relevance since the experiments were performed in a buffer more similar to physiological conditions.

Prolonged survival of corneal allografts incubated in alloantibody fragments.

In this study, we determined the binding characteristics of F(ab')2 alloantibody fragments to corneal antigens and assessed the capacity of these antibody fragments to protect corneal allografts from immune attack. Goat anti-rabbit alloantibodies were pepsin-digested and labeled with 125I, and the time course of association and dissociation of the F(ab')2 fragments was determined. Corneal allografts were incubated in unlabeled F(ab')2 fragments and transplanted into allogeneic recipients, and the graft survival times were recorded. Binding of radiolabeled F(ab')2 fragments to rabbit cornea cells reached a maximum at 12 hr. At 32 degrees C (rabbit corneal temperature), the radiolabel eluted rapidly from the cornea, reaching baseline at 72 hr. At 4 degrees C (corneal graft storage temperature), significant amounts remained associated with the cornea at 96 hr. Mean survival time for grafts incubated in F(ab')2 anti-rabbit fragments was significantly greater than that of grafts incubated in nonimmune F(ab')2 fragments. Three of the corneal allografts incubated in goat F(ab')2 anti-rabbit fragments survived for 100 days, whereas the longest surviving control allograft incubated in goat F(ab')2 nonimmune fragments was rejected on day 24. Preincubation of corneas in unlabeled, immune F(ab')2 fragments followed by incubation in radiolabeled, immune F(ab')2 fragments suggested that antigen masking was not a factor in the prolongation of graft survival. Based on the binding and release kinetics and the graft survival times, it appears that the protective effect of immune F(ab')2 fragments extends well beyond the binding interval of the antibody fragments to corneal cell membranes.

Fiber Optic Biosensor for Cyclodiene Insecticides

Chlorendic caproic acid (CCA) was used to synthesize hexachlorocyclopentadienefluorescein (FL) and bovine serum albumin (BSA) conjugates. Anti-CCA antibodies (CCA−Abs), which were raised against BSA−CCA and immobilized on quartz fibers, bound FL−CCA selectively and reversibly. Fluorescence generated by evanescent excitation of the bound FL−CCA was used to monitor the binding event. The affinity of CCA−Abs for FL−CCA (KD = 1.9 nM) was calculated from the time courses of association and dissociation of FL−CCA. The cyclodiene insecticides chlordane, heptachlor, dieldrin, endrin, aldrin, and endosulfan competed with FL−CCA for binding to CCA−Abs and reduced fluorescence in a concentration-dependent manner with the following rank order: chlordane > heptachlor > dieldrin > aldrin > endosulfan. This fiber optic fluoroimmunosensor detects cyclodiene insecticides at the ppb level, has low cross-reactivity with γ-hexachlorocyclohexane, and does not detect (p,p'-dichlorodiphenyl)trichloroethane (DDT). Keywords: Cyc...

Mineralocorticoid (Type I) Receptors in the Olfactory Mucosa of the Mammal: Studies with [3H]Aldosterone and the Anti-mineralocorticoid Spironolactone

High-affinity, specific binding sites to mineralocorticoids, with characteristics of mineralocorticoid (type I) receptors, have been found in the mammalian olfactory mucosa. In the presence of RU 28362, which blocks low-affinity binding of the labeled hormone to glucocorticoid (type II) receptors, Scatchard analysis of the specific [3H]aldosterone binding indicates that aldosterone binds to a single class of high-affinity (type I) sites with a dissociation constant, Kd, of 1.2 x 10(-9) M and a maximum number of binding sites, Bmax, of 63 fmol/mg dry tissue. Time course of association of [3H]aldosterone and its binding sites showed maximal binding by 30 min at 25 degrees C which remained unchanged up to 90 min. Competition studies performed with 1 x 10(-7)-1 x 10(-5) M spironolactone, a competitive mineralocorticoid antagonist, showed a dose-response reduction of [3H]aldosterone binding, implying that in the olfactory mucosa this compound behaves as an aldosterone antagonist. These results demonstrate significant [3H]aldosterone binding to receptors of high affinity and mineralocorticoid specificity and suggest that the olfactory mucosa is a target site for mineralocorticoid action.

Kinetic analysis of ligand-receptor association data that display an overshoot phenomenon.

A binding overshoot was frequently observed in the time course of association of diazepam with rat brain membrane receptors shortly after the start of the interaction. Such time profiles most likely reflect the "receptor switch" mechanism, assuming an equilibrium between two forms of a receptor (R and R*) that possess different affinities to the ligand (L) in question. Similar effects could be caused by the presence of a slowly dissociating competitor. The kinetics of these mechanisms were verified by simulation of theoretical time courses. A computer program for simulation of the time course, and estimation of rate constants of the individual reaction steps, was developed and is described in this communication. It employs the Euler-Cauchy integration for simulation of theoretical time courses. Optimised estimates of the rate constants were computed by simultaneous random variation of parameters within a pre-set interval. Stable solutions can be obtained for this system, thus enabling evaluation of equilibrium constants defined by the model. The source code is available in Turbo-Pascal. It can be used, after re-writing the rate equations, for fitting of similar kinetic models to suitable experimental data.

Comparative kinetic study of the binding between 2,3,7,8‐tetrachlorodibenzo‐ρ‐dioxin and related ligands with the hepatic Ah receptors from several rodent species

Kinetic analysis of the time course of association of [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin with hepatic cytosol from five rodent species gave additional evidence for differences in the properties of the Ah receptor ligand binding subunit between species. A parallel study of the association of six tritiated polychlorinated dibenzo-p-dioxins and dibenzofurans with hepatic Ah receptor from Wistar rat and C57BL/6 mouse showed that their rank order for kinetic affinity did not correlate with the rank ordering of their toxic potency and may vary according to the source of the Ah receptor.

MONOCLONAL-ANTIBODIES TO HUIFN-ALPHA SPECIES APPLIED TO THE STUDY OF IFN-RECEPTOR INTERACTION INDICATE THAT RECEPTOR DOWN-REGULATION DOES NOT OCCUR IN DAUDI CELLS

Monoclonal antibodies to HuIFN- alpha species were previously isolated and the reactive epitopes on HuIFN- alpha2 identified. When used in excess concentration the antibodies neutralise the antiviral activity of HuIFN- alpha and inhibit binding of IFN to its receptor on human and bovine cells. However, when used in equimolar concentrations the antibodies bind to IFN bound to its receptor on human but not on bovine cells. Radiolabelled antibodies and radiolabelled ligand have been used to study in detail IFN-receptor interactions in human cells. The use of the monoclonal antibodies has identified differences in the interaction in different cell lines not obvious from ligand binding studies alone. IFN receptors do not appear to be down-regulated in Daudi cells but remain on the cell surface complexed with bound IFN whereas down-regulation does occur in the human epithelial breast carcinoma cell line BT20. Although surface receptors are not decreased in Daudi cells exposed to IFN, the time course of association of radiolabelled IFN with these cells shows that cell associated radioactivity decreases after incubation for more than 1 hr at 37-degrees-C. This result cannot be explained by degradation of exogeneous IFN (which does not occur) and suggests that endogenous IFN may be involved in the interaction of HuIFN- alpha with Daudi cells.

Evidence for two sites on rat liver plasma membranes which interact with high density lipoprotein.

There is little dispute that high density lipoprotein (HDL) binds to cells, however, the nature of the interaction is not fully understood. We now present evidence for a new binding site of higher affinity but lower capacity than the sites previously described in the literature. This new site is characterized by high affinity/low capacity for HDL binding (Kd = 0.94 microgram/ml, Bmax = 36 ng/mg), while the low affinity site (Kd = 36 micrograms/ml, Bmax approximately 700 ng/mg) appears to be consistent with the literature values for the interaction of HDL with cells and isolated membranes. Proteolysis of HDL with trypsin abolished its interaction with the high affinity site, suggesting an apolipoprotein requirement, while having no effect on binding to the lower affinity site. Kinetic rates of association/dissociation were determined in order to further characterize the high affinity site. At a concentration which favored the binding of HDL with the high affinity site (1 microgram/ml, 37 degrees C), the time course of association of HDL with rat liver plasma membranes, displayed a biphasic pattern, requiring 6-8 h to reach the level of binding predicted from the saturation studies. The second phase was highly sensitive to temperature, being considerably slower at 24 degrees C and totally abolished at 0 degrees C. A kinetic Kd, derived from the measured association and dissociation rate constants (Kd = 0.31 microgram/ml), was found to be of a similar magnitude to the Kd calculated for the high affinity site by Scatchard analysis (Kd = 0.94 microgram/ml). In summary, the high affinity site on rat liver plasma membranes displays an apoprotein requirement and kinetic parameters, consistent with a ligand-receptor interaction.

High-affinity phlorizin binding in Mytilus gill

The gill of the marine mussel, Mytilus, contains a high affinity, Na-dependent d-glucose transporter capable of accumulating glucose directly from sea water. We examined the ability of the β-glucoside, phlorizin, to act as a high-affinity ligand of this process in intact gills and isolated brush border membrane vesicles (BBMV). The time course of association of nanomolar [ 3H]phlorizin to gills and BBMV was slow, with t 50 values between 10 and 30 min, and a half-time for dissociation of approx. 30 min. 1 mM d-glucose reduced equilibrium binding of 1 nM phlorizin by 90–95%, indicating that there was little non-specific binding of this ligand to the gill. In addition, there was little, if any hydrolysis by the gill of phlorizin to its constituents, glucose and phloretin. Phlorizin binding to gills and BBMV was significantly inhibited by the addition of 50 μM concentrations of d-glucose and α-methyl- d-glucose, and unaffected by the addition of l-glucose and fructose. Binding to gills and BBMV was reduced by > 90% when Na + was replaced by K +. Replacement of Na + by Li + effectively blocked binding to the intact gill, although Li + did support a limited amount of glucose-specific phlorizin binding in BBMV. The K d values for glucose-specific phlorizin binding in intact gills and BBMV were 0.5 nM and 6nM, respectively. We conclude that phlorizin binds with extremely high affinity to the Na-dependent glucose transporter of Mytilus gill, which may be useful in future efforts to isolate and purify the protein(s) involved in integumental glucose transport.

Characterization of the angiotensin II receptor in primary cultures of rat hepatocytes. Evidence that a single population is coupled to two different responses.

The angiotensin II receptor of cultured rat hepatocytes was characterized using [3H]angiotensin II as radioligand. Binding at 23 degrees C was rapid (t1/2 = 0.65 min) with equilibrium being reached in 10-12 min. At this time, binding was completely reversible after 20 min (t1/2 = 3.5 min), indicating negligible internalization of the ligand. Analysis of the saturation binding curve showed one population of binding sites with an apparent KD of 8.6 nM and a Bmax of 35 fmol/mg of protein. The time courses of association and dissociation were also consistent with one class of binding sites with an apparent kinetically derived KD of 7.7 nM. The order of potency of different agonists and antagonists to increase cytosolic Ca2+ or phosphorylase a or inhibit the effects of angiotensin II on these parameters was the same as for their mimicry or reversal of angiotensin II inhibition of glucagon-induced cAMP accumulation, and was well correlated with their order of potency to inhibit angiotensin II specific binding. Treatment of cultured hepatocytes with dithiothreitol caused a time- and concentration-dependent inhibition of angiotensin II binding and corresponding alterations of angiotensin II effects on phosphorylase and cAMP. It also inhibited the actions of other hormones on phosphorylase. These results indicate that hepatocytes contain a homogeneous population of angiotensin II receptors that are coupled to two different biological effects apparently mediated by different G-proteins.

Does the prolonged occupancy of M1 receptors by telenzepine protect them against the action of vagally released acetylcholine?

The affinities of telenzepine, pirenzepine and atropine for muscarinic acetylcholine receptors have been determined by receptor binding studies using brain cortex and heart membranes of calf and rat. The ratios of affinities of telenzepine and pirenzepine for the cortical M1 receptors, cortical 'non-M1' receptors and cardiac M2 receptors are 50:5:1 and 80:5:1, respectively. The time course of association of telenzepine and pirenzepine with M1 receptors is similar, whereas the half-times for dissociation are 35 and 2.3 min at 37 degrees C, respectively. In further experiments this slow dissociation rate of telenzepine is shown to be rate-limiting for the occupation of M1 receptors by other ligands. The prolonged occupation of M1 receptors by telenzepine is discussed as a possible protective mechanism against acetylcholine released from vagal nerve fibers.

The Endothelial Cell and the Factor VIII Bypassing Activity of Prothrombin Complex Concentrate

Some classical hemophiliacs have a paradoxical hemostatic response to prothrombin complex concentrate (PCC). We hypothesized that vascular endothelial cells (EC) may contribute to this "factor VIII bypassing activity". When PCC were incubated with suspensions or monolayer cultures of EC, they acquired the ability to partially bypass the defect of factor VIII deficient plasma. This factor VIII bypassing activity distributed with EC and not with the supernatant PCC, and was not a general property of intravascular cells. The effect of PCC was even more dramatic on fixed EC monolayers, which became procoagulant after incubation with PCC. The time courses of association and dissociation of the PCC-derived factor VIII bypassing activity of fixed and viable EC monolayers were both rapid. We conclude that EC may provide a privileged site for sequestration of constituents of PCC which express coagulant activity and which bypass the abnormality of factor VIII deficient plasma.

Pharmacologic relevance of dihydropyridine binding sites in membranes from rat aorta: kinetic and equilibrium studies.

The kinetic features of the interaction of dihydropyridines with rat aortic smooth muscle were investigated in parallel mechanical and binding studies. The inhibitory action of (+)-PN200-110 and nisoldipine on contractions evoked by potassium chloride depolarization was characterized by a pronounced time dependency, in which the inhibition increased slowly after depolarization to attain a steady-state value, while with (-)-PN200-110 and (+)-Bay K8644 the inhibition was almost instantaneous. To explain these observations, specific binding sites for dihydropyridines were studied in membranes isolated from rat aorta, using [3H](+)-PN200-110 as a radioligand. We found that the time course of the development of inhibition of potassium chloride-evoked contractions by various concentrations of (+)-PN200-110 paralleled the time course of [3H](+)-PN200-110 binding to isolated membranes and that the level of inhibition was predictable from the level of occupation of these binding sites. These results indicate that depolarization increases the affinity of calcium channels for dihydropyridines in vascular smooth muscle and that the time course of the inhibitory effect on contraction is determined by the time course of association with the high-affinity state of the channel.

Active form of Ca2+-dependent phosphatidylinositol phospholipase C from rat lymphocytes at physiological Ca2+ concentrations

at equilibrium was competitively inhibited by somatostatin14, with an ICs,, of 0.22 _+ 0.06 (3) nM. Scatchard analysis of the data (Fig. la) revealed the presence of a single class of sites with a k’, of 0.19 f 0.05 (3)nM and a B,,, of 12.8 f 1.8 (3)fmolimg of protein. The time course of association of H-somatostatin closely resembled that of I” I-Tyr” -somatostatin. equilibrium being attained within 20 min of incubation. Specific binding at equilibrium represented 33. I % of the total bound ligand. The association rate constant was 1.6 x 10‘ M ’ s I. ‘H-somatostatin bound to the cells was displaced by increasing concentrations of somatostatin-14, with an IC,,] of 3.48 f 0.24 (3)nM. However, Scatchard analysis of the data (Fig. 13) gave a curvilinear plot which, on fitting to a two-site model, gave the following parameters: Kdl 0.29 f 0.06 (3) nM; B,,, , 40.4 f 14.2 (3)fmol/mg of protein; K,, 15.43 f 5.96 (3) nM; B,,, 205.3 & 52.7 (3) fmol/mg of protein. Further evidence for the presence of two sites was obtained using SMS 201-995. which displaced only 50% of bound ’ H-somatostatin. Although ’” I-Tyr” -somatostatin revealed one class of binding sites on neuroblastoma x glioma 108CC15 cells, the curvilinearity of the Scathard plot obtained using ‘Hsomatostatin suggested the presence of two classes of sites with affinities not significantly different from those found in the rat brain using the same ligand (Weightman ef a!., 1985). The partial displacement of somatostatin binding by SMS 201-995 provides further evidence for the existence of two distinct subpopulations of binding sites, in agreement with the findings in rat brain (Reubi, 1984; C. A. Whitford, unpublished work). Thus the close similarities in receptor characteristics between the rat brain and 108CC15 cells, using H-somatostatin, makes these cells a potential model system for the study of the physiological control of somatostatin receptors and their second messenger systems.