Interaction of hepatic asialoglycoprotein receptor with asialoorosomucoid and galactolyzed lysosomal α-glucosidase

An asialoglycoprotein receptor was isolated from murine liver and purified more than 1600-fold using 2-fold affinity chromatography on asialoorosomucoid-Sepharose. The purified receptor did not interact with ¹²⁵I-orosomucoid, but bound to ¹²⁵I-asialoorosomucoid. The binding of the receptor to asialoorosomucoid was saturable. The dissociation constant of the receptor-asialoorosomucoid complex was 0.4·10⁻⁹ M. The molecular mass of the receptor, as determined with the use of specific antibodies by the immunoblotting method, was 43 kDa. High concentrations of unlabeled asialoorosomucoid and of n-aminophenyl-β-d-galactosyl derivatives of bovine serum albumin, ovalbumin and acid α-glucosidase from human liver inhibited the binding of the receptor to ¹²⁵I-asialoorosomucoid almost completely. The binding of the receptor to ¹²⁵I-galactolyzed α-glucosidase was pH-dependent, with the pH optimum at 8.0–9.0. It was shown that, as in the case of ¹²⁵I-asialoorosomucoid, the binding of the ¹²⁵I-galactosyl derivative of α-glucosidase occurred in the presence of Ca²⁺ and was inhibited by N-acetylgalactosamine. Glycoproteins containing galactose as a terminal residue inhibited the interaction of the receptor with ¹²⁵I-galactolyzed α-glucosidase. The possibility of directed transport of the galactolyzed α-glucosidase derivative into parenchymous liver cells using receptor-mediated endocytosis is discussed.     

A quantitative analysis of glucagon binding to isolated intact neonatal and adult rat hepatocytes on the basis of two different binding models

The interaction of glucagon with specific receptors has been studied in isolated intact neonatal and adult rat hepatocytes. The hormone binding measured directly with ¹²⁵I-labelled glucagon was saturable and reversible. The ¹²⁵I-labelled glucagon binding was inhibited by unlabelled homologous hormone at concentrations ranging from 0.5 nM to 50 μM. Two different binding models were assumed to analyse the binding data by a nonlinear least-squares procedure: (I) a single class of independent sites and (II) two classes of independent sites. The comparison of the fitted theoretical curves reveals that both binding models are in fact compatible with these data. Adult hepatocytes have a considerably higher affinity for glucagon than neonatal hepatocytes; the binding capacity of neonatal liver cells from 1–7-days-old rats proved to be markedly reduced compared with the cells from adult rats. The glucagon-induced intracellular cyclic AMP production was measured at various hormone concentrations under conditions identical to those for the determination of extracellular hormone binding. The correlation of both parameters indicates a direct connection between receptor-occupancy and adenylate cyclase stimulation. These results suggest that a decrease receptor concentration in neonatal hepatocytes is responsible for the decreased cyclic AMP production.     

Galactosyl transferase-the liver plasma membrane binding-site for asialo-glycoproteins

Agalacto-fetuin inhibits the binding of ¹²⁵I-asialo-fetuin by liver plasma membrane fragments. The chemically prepared agalacto-glycoprotein derivative is not a substrate for plasma membrane sialyl transferase and therefore this indicates that agalacto-fetuin is a true inhibitor of the membrane binding of ¹²⁵I-asialo-fetuin. The plasma membrane fraction also contains galactosyl transferase activity and the binding of ¹²⁵I-asialo-fetuin by plasma membranes is prevented by α-lactalbumin, a known inhibitor of glycoprotein-galactosyl transferase. These data indicate that galactosyl transferase is the liver plasma membrane component which binds asialo-glycoproteins.     

Structural Characterization by Affinity Cross-Linking of Glucagon-Like Peptide-1(7–36)Amide Receptor in Rat Brain

Abstract: Specific binding of glucagon-like peptide (GLP)-1(7–36)amide was detected in several rat brain areas, with the highest values being found in hypothalamic nuclei and the nucleus of the solitary tract. In hypothalamus and brainstem homogenate binding of ¹²⁵I-GLP-1(7–36)amide was time, temperature, and protein content dependent and was inhibited by unlabeled proglucagon-derived peptides. The rank order of potency was GLP-1(7–36)amide ? GLP-1(1–36)amide > GLP-1(1–37) ? GLP-2 > glucagon. Scatchard analysis of the steady-state binding data was consistent with the presence of both high- and low-affinity binding sites in hypothalamus and brainstem. Brain ¹²⁵I-GLP-1(7–36)amide-binding protein complexes were covalently cross-linked using disuccinimidyl suberate and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single radiolabeled band of M_r 56,000 identified in both hypothalamus and brainstem homogenates was unaffected by reducing agents. An excess of unlabeled GLP-1(7–36)amide abolished the band labeling, whereas glucagon had no effect. Other unlabeled GLPs inhibited M_r 56,000 complex labeling with the following order of potency: GLP-1(1–36)amide > GLP-1(1–37) > GLP-2. The binding of ¹²⁵I-GLP-1(7–36)amide and the intensity of the cross-linked band were similarly inhibited in a dose-response manner by increasing concentrations of unlabeled GLP-1(7–36)amide. Covalent M_r 56,000 ¹²⁵I-GLP-1(7–36)amide-binding protein complexes solubilized by Triton X-100 were adsorbed onto wheat germ agglutinin. Our results suggest that the GLP-1(7–36)amide receptor in rat brain is a glycoprotein with a single binding subunit that has a greater molecular weight but binding features and ligand specificity similar to those of its peripheral tissue counterparts.     

Structure:function studies of receptors for thyrotropin and tetanus toxin: lipid modulation of effector binding to the glycoprotein receptor component.

¹²⁵I-labeled tetanus toxin interacts with the glycoprotein component of the thyroid thyrotropin receptor when this component is in solution or when it is incorporated into a liposome. Binding can be inhibited by both unlabeled thyrotropin and tetanus toxin but not by unlabeled prolactin, glucagon, insulin, ACTH, or growth hormone; binding can also be inhibited by a purified fragment of the glycoprotein component of the receptor. Changing the phospholipid of the liposome matrix from dipalmitoyl phosphatidylcholine to dioleoyl phosphatidylcholine significantly increases the binding of ¹²⁵I-TSH to the glycoprotein component of the receptor but does not affect ¹²⁵I-tetanus toxin binding.     

Studies of albumin binding to rat liver plasma membranes: Implications for the albumin receptor hypothesis

To characterize a previously proposed hepatocyte albumin receptor, we examined the binding of native and defatted ¹²⁵I-labeled rat albumin to rat liver plasma membranes. After incubation for 30 min, binding was determined from the distribution of radioactivity between membrane pellet and supernatant following initial centrifugation (15 000 × g for 15 min), after repeated cycles of washing with buffer and re-centrifugation. ¹²⁵I-labeled albumin recovered in the initial membrane pellet averaged only 4% of that incubated. Moreover, this albumin was only loosely associated with the membrane, as indicated by recovery in the pellet of under 0.5% of the counts after three washes. Binding of ¹²⁵I-labeled albumin to the plasma membranes was no greater than to erythrocyte ghosts, was not inhibited by excess unlabeled albumin, and was not decreased by heat denaturation of the membranes, all suggestive of a lack of specific binding. Failure to observe albumin binding to the membranes was not due to a rapid dissociation rate or ‘off-time’, as incubations in the presence of sufficient ultraviolet light to promote covalent binding of ligands to receptors did not increase ¹²⁵I counts bound to the membrane. Finally, affinity chromatography over albumin/agarose gel of solubilized membrane proteins provided no evidence of a membrane protein with a high affinity for albumin. These studies, therefore, do not support the hypothesis that liver cell plasma membranes contain a specific albumin receptor.     

Characterization of specific binding sites for vasoactive intestinal peptide in rat intestinal epithelial cell membranes

Specific binding sites for vasoactive intestinal peptide were characterized in plasma membranes from rat intestinal epithelial cells. At 30°C, the interaction of ¹²⁵I-labelled peptide with intestinal membranes was rapid, reversible, specific and saturable. At equilibrium, the binding of ¹²⁵I-labelled peptide was competitively inhibited by native peptide in the 3 · 10^?11?3 · 10^?7 M range concentration. Scatchard analysis of binding data suggested the presence of two distinct classes of vasoactive intestinal peptide binding sites: a class with a high affinity K_d = 0.28 nM) and a low capacity (0.8 pmol peptide/mg membrane protein) and a class with a low affinity (K_d = 152 nM) and a high capacity (161 pmol peptide/mg membrane protein). Secretin competitively inhibited binding of ¹²⁵I-labelled peptide but its potency was 1/1000 that of native peptide. Glucagon and the gastric inhibitory peptide were ineffective. The guanine nucleotides, GTP and Gpp(NH)p inhibited markedly the interaction of ¹²⁵I-labelled peptide with its binding sites, by increasing the rate of dissociation of peptide bound to membranes. The other nucleotides triphosphate tested (ATP, ITP, UTP, CTP) were also effective in inhibiting binding of ¹²⁵I-labelled peptide to membranes but their potencies were 1/100-1/1000 that of guanine nucleotides.The specificity and affinity of the vasoactive intestinal peptide-binding sites in plasma membranes prepared from rat intestinal epithelial cells, which is in agreement with an adenylate cyclase highly sensitive to the peptide recently characterized in these membranes (Amiranoff, B., Laburthe, M., Dupont, C. and Rosselin, G. (1978) Biochim. Biophys. Acta 544, 474–481) further argue for a physiological role of the peptide in the regulation of intestinal epithelial function.     

Binding and degradation of 125I-glucagon by highly purified rat liver plasma membranes

¹²⁵I-glucagon binding and degradation were studied in highly purified plasma membranes from rat livers. Specific ¹²⁵I-glucagon binding increased rapidly with time at 30°C and reached a maximum between 30 and 120 min. At 120 min the labelled material present in the supernatants from incubation mixtures had extensively lost its ability to rebind to fresh membranes whatever the glucagon concentration. This impairment was not due to the release of a degradative activity into the incubation mixture, suggesting a membrane-mediated process. The presence of proteinase inhibitors (bacitracin/aprotinin) resulted both in an increase in specific ¹²⁵I-glucagon binding to membranes and an improvement in the ability of the labelled material from the supernatant to rebind to fresh membranes. When analysed by Bio-Gel P-10 chromatography the loss in the ability of the labelled material in the supernatants to rebind to fresh membranes correlated with a decrease in the labelled material which eluted as ¹²⁵I-glucagon from the column. Chromatographic analysis overestimated ¹²⁵I-glucagon when compared to the radioreceptor assay. The labelled material extracted from membranes by Triton X-100 solubilization or dissociated from membranes after exposure to an excess of unlabelled glucagon mainly eluted as ¹²⁵I-glucagon. However, a significant amount (20–30%) of the labelled material eluted in the low molecular weight region.     

Receptors for Vasoactive Intestinal Polypeptide on Isolated Synaptosomes from Rat Cerebral Cortex. Heterogeneity of Binding and Desensitization of Receptors

Abstract: Vasoactive intestinal polypeptide (VIP) is a neuropeptide that causes neurone excitation in the brain cortex. VIP receptors were studied in subcellular fractions isolated from rat cerebral cortex. The receptor binding of ¹²⁵I-VIP was greatest in the synaptosomal fraction at membrane protein concentrations of 50–100 μg/ml, a temperature of 37°C, and a pH from 7.4 to 7.7. Under these conditions the concomitant proteolytic degradation of ¹²⁵I-VIP was approximately 10% after 60 min of incubation. The binding of 60 pmoI/L ¹²⁵I-VIP reached steady-state after 60 min and was maintained up to 240 min. At steady-state, the receptor-bound 125I-VIP was displaced by unlabelled VIP with half-maximal inhibition (IC₅₀) at a concentration of approximately 3 nmol/L. The binding of ¹²⁵I-VIP in the concentration range of 10 pmol/L to 6 nmol/L was superimposable on the VIP displacement curve. The Scatchard plot was curvilinear with upward concavity, which can be interpreted to represent two classes of receptors with K_D of 2.5 and 125 nmol/L, one class of receptors with negative cooperative interactions, or heterogeneity of the ¹²⁵I- VIP preparation. The total amount of receptors was 9.5 pmol/mg of membrane protein. Secretin displaced receptor-bound 125I-VIP with an IC₅₀ of 0.3 μmol/L, whereas glucagon snowed no inhibition up to 1 μmol/L. The dissociation of receptor-bound ¹²⁵I-VIP was biexponential with rate constants (k₂) of 4.1 – 10^?3 and 0.18 min^?1 corresponding to half-times of approximately 170 and 4 min, respectively. The size of the two components was dependent on the duration of the ¹²⁵I-VIP association period. Initially, both components increased; at steady-state, the rapid component declined, whereas the slow component increased to approximately 70% after 120 min. The association rate constants (k₁) were estimated from the initial velocities as 10⁶ and 4. 10⁶ L. mol^?1. min^?1, and a calculation of the K_D as k₂/k₁ gave values of 4.1 and 45 nmol/L, respectively. In conclusion, the presence of receptors for VIP on synaptosomes from the cerebral cortex supports the role of VIP as a neurotransmitter in the brain. The receptor binding was heterogeneous, suggesting the presence of two classes of receptors. The binding kinetics showed a time-dependent transition of VIP receptors from a low- to a high-affinity state, which may be interpreted as desensitisation of synapses to the action of VIP.     

Interaction of Cyclic AMP Derivatives with the Angiotensin II Receptor

Abstract

The effect of cyclic AMP and of its derivatives was studied on ¹²⁵I-angiotensin II and ¹²⁵I- (Sar¹, Ala⁸) -angiotensin II binding to rat adrenal membrane receptors. Dibutyryl cyclic AMP, 8-bro-mo-cyclic AMP and cyclic AMP inhibited both agonist and antagonist binding in a specific and dose-dependent way, with K₁ of 1.3 mM, 6.8 mM and about 30 mM, respectively. Scatchard analysis of binding data indicated that the nucleotides interacted directly with the membrane receptor for angiotensin II. These results suggest that cyclic AMP may act extracellularly and affect receptor-mediated events.     

Interaction of lipophorin with Rhodnius prolixus oocytes: biochemical properties and the importance of blood feeding

Lipophorin (Lp) is the main haemolymphatic lipoprotein in insects and transports lipids between different organs. In adult females, lipophorin delivers lipids to growing oocytes. In this study, the interaction of this lipoprotein with the ovaries of Rhodnius prolixus was characterised using an oocyte membrane preparation and purified radiolabelled Lp ⁽¹²⁵I-Lp). Lp-specific binding to the oocyte membrane reached equilibrium after 40-60 min and when ¹²⁵I-Lp was incubated with increasing amounts of membrane protein, corresponding increases in Lp binding were observed. The specific binding of Lp to the membrane preparation was a saturable process, with a K_dof 7.1 ± 0.9 x 10^-8M and a maximal binding capacity of 430 ± 40 ng ¹²⁵I-Lp/µg of membrane protein. The binding was calcium independent and pH sensitive, reaching its maximum at pH 5.2-5.7. Suramin inhibited the binding interaction between Lp and the oocyte membranes, which was completely abolished at 0.5 mM suramin. The oocyte membrane preparation from R. prolixus also showed binding to Lp from Manduca sexta. When Lp was fluorescently labelled and injected into vitellogenic females, the level of Lp-oocyte binding was much higher in females that were fed whole blood than in those fed blood plasma.     

Synthesis and properties of a photoaffinity probe for the glucagon receptor in hepatocyte plasma membranes

The reaction of glucagon with 4-fluoro-3-nitrophenylazide has been shown to afford the photosensitive derivative, N^?-4-azido-2-nitrophenyl-glucagon. The structure and properties of this derivative were established by amino acid analysis, absorption and fluorescence spectroscopy, deamination, Edman degradation and photolysis. This photoaffinity derivative of glucagon has been used to label specifically glucagon binding sites on hepatocyte plasma membranes.     

125I-Ifenprodil: Synthesis and Characterization of Binding to a Polyamine-Sensitive Site in Cerebral Cortical Membranes

Abstract: The characteristics of binding sites in rat cerebral cortical synaptic membranes labeled by ¹²⁵I-ifenprodil, a noncompetitive NMDA receptor antagonist, are described. ¹²⁵I-ifenprodil was synthesized using Na¹²⁵I in the presence of chloramine-T and purified by paper chromatography. Binding of the ¹²⁵I-ligand was optimal at pH 7.7 in 5 mM Tris · HCl buffer. Equilibrium binding of ¹²⁵I-ifenprodil was displaced by spermine (1 mM) but not by ifenprodil or its analogue, SL 82.0715 (both 16.7 μM). Zn²⁺, Ca²⁺, and Mg²⁺ inhibited specific binding of ¹²⁵I-ifenprodil in a concentration-dependent manner, with IC₅₀ values of 0.11, 1.1, and 1.7 mM, respectively. The dissociation constant (K_D) for unlabeled ifenprodil determined by saturation binding was 205 nM. Scatchard plots of saturation data appeared curvilinear but were best described by a single-binding-site model (Hill coefficient = 0.95), with a density of binding sites (B_max) of 141 pmol/mg of protein. Binding of ¹²⁵I-ifenprodil was inhibited by polyamines, with a rank potency order of spermine > spermidine > putrescine = 1,3-diaminopropane. The pattern of inhibition produced by spermidine was apparently competitive. Ifenprodil congeners also fully inhibited polyamine-sensitive binding of ¹²⁵I-ifenprodil, with a rank potency order of ifenprodil > SL 82.0715 = tibalosine > nylidrin = isoxsuprine. It was found that σ/antitussive agents partially inhibited specific binding, but inclusion of the σ drug GBR 12909 had little effect on the binding of ¹²⁵I-ifenprodil, suggesting this site was not involved. The binding site labeled by ¹²⁵I-ifenprodil is polyamine sensitive, has a discrete pharmacological profile, and apparently is unrelated to the σ site.     

Binding and degradation of125I-insulin by isolated rat renal brush border membranes: Evidence for low affinity,high capacity insulin recognition sites

Summary The kidney plays a major role in the handling of circulating insulin in the blood, primarily via reuptake of filtered insulin at the luminal brush border membrane.¹²⁵I-insulin associated with rat renal brush border membrane vesicles (BBV) in a time-and temperature-dependent manner accompanied by degradation of the hormone to trichloroacetic acid (TCA)-soluble fragments. Both association and degradation of¹²⁵I-insulin were linearly proportional to membrane protein concentration with virtually all of the degradative activity being membrane assoicated. Insulin, proinsulin and desoctapeptide insulin all inhibited the association and degradation of¹²⁵I-insulin by BBV, but these processes were not appreciably afected by the insulin-like growth factors IGF-I and IGF-II or by cytochromec and lysozyme, low molecular weight, filterable, proteins, which are known to be reabsorbed in the renal tubules by luminal endocytosis. When the interaction of¹²⁵I-insulin with BBV was studied at various medium osmolarities (300–1100 mosm) to alter intravesicular space, association of the ligand with the vesicles was unaffected, but degradation of the ligand by the vesicles decreased progressively with increasing medium osmolarity. Therefore, association of¹²⁵I-insulin to BBV represented binding of the ligand to the membrane surface and not uptake of the hormone or its degradation products into the vesicles. Attempts to crosslink¹²⁵I-insulin to a high-affinity insulin receptor using the bifunctional reagent disuccinimidyl suberate revealed only trace amounts of an¹²⁵I-insulin-receptor complex in brush border membrane vesicles in contrast to intact renal tubules where this complex was readily observed. Both binding and degradation of¹²⁵I-insulin by brush border membranes did not reach saturation even at concentrations of insulin approaching 10^–5 m. These results indicate the presence of low-affinity, high-capacity binding sites for¹²⁵I-insulin on renal brush border membranes which can clearly distinguish insulin from the insulin-like growth factors and other low molecular weight proteins and polypeptides, but which do not differentiate insulin from its analogues ad do the biological receptors for the hormone. The properties and location of these binding sites make them attractive candidates for the sites at which insulin is reabsorbed in the renal tubule.     

Interaction of Bacillus thuringiensis Toxins with Larval Midgut Binding Sites of Helicoverpa armigera (Lepidoptera: Noctuidae)

In 1996, Bt-cotton (cotton expressing a Bacillus thuringiensis toxin gene) expressing the Cry1Ac protein was commercially introduced to control cotton pests. A threat to this first generation of transgenic cotton is the evolution of resistance by the insects. Second-generation Bt-cotton has been developed with either new B. thuringiensis genes or with a combination of cry genes. However, one requirement for the “stacked” gene strategy to work is that the stacked toxins bind to different binding sites. In the present study, the binding of ¹²⁵I-labeled Cry1Ab protein (¹²⁵I-Cry1Ab) and ¹²⁵I-Cry1Ac to brush border membrane vesicles (BBMV) of Helicoverpa armigera was analyzed in competition experiments with 11 nonlabeled Cry proteins. The results indicate that Cry1Aa, Cry1Ab, and Cry1Ac competed for common binding sites. No other Cry proteins tested competed for either ¹²⁵I-Cry1Ab or ¹²⁵I-Cry1Ac binding, except Cry1Ja, which competed only at the highest concentrations used. Furthermore, BBMV from four H. armigera populations were also tested with ¹²⁵I-Cry1Ac and Cry1Ab to check the influence of the insect population on the binding results. Finally, the inhibitory effect of selected sugars and lectins was also determined. ¹²⁵I-Cry1Ac binding was strongly inhibited by N-acetylgalactosamine, sialic acid, and concanavalin A and moderately inhibited by soybean agglutinin. In contrast, ¹²⁵I-Cry1Ab binding was only significantly inhibited by concanavalin A. These results show that Cry1Ac and Cry1Ab use different epitopes for binding to BBMV.     

Identification of a crotoxin-binding protein in membranes from guinea pig brain by photoaffinity labeling

Crotoxin is a neurotoxic phospholipase A₂ capable of blocking synaptic transmission by inhibiting the release of neurotransmitters. The photoaffinity labeling technique was used to identify the neural membrane molecules involved in the binding of crotoxin. A photoactivatable, radioactive derivative of crotoxin was synthesized by reacting crotoxin withN-hydroxysuccinimidyl-4-azidobenzoate and with Na[¹²⁵I]. Photoirradiation of synaptosomes from guinea pig brains in the presence of the crotoxin derivative resulted in the formation of a major radioactive conjugate of 100,000 daltons as revealed by autoradiography of a sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern. Pretreatment of the synaptosomes with trypsin,Staphylococcus aureus protease, or papain prevented the formation of this conjugate. The conjugate was not detected when plasma membranes from several nonneural tissues replaced the brain synaptosomes. Unmodified crotoxin inhibited the formation of this adduct with an IC₅₀ of about 10^–8 M. Mojave toxin, caudoxin, notexin,Naja naja PLA, and taipoxin also inhibited adduct formation with different potencies, while -bungarotoxin and pancreatic PLA were ineffective. We concluded that an 85,000-dalton protein is the major component responsible for the binding of crotoxin to synaptosomal membranes.On leave from Department of Biochemistry and Biophysics, University of Hawaii School of Medicine, Honolulu, Hawaii.     

Preparation of 125I-calmodulin with retention of full biological activity: Its binding to human erythrocyte ghosts

Of several methods employed for preparing ¹²⁵I-calmodulin, only the glucose oxidase-lactoperoxidase system under controlled conditions produced an iodinated derivative which retained complete biological activity. Unlabeled calmodulin and ¹²⁵I-calmodulin stimulated cyclic nucleotide phosphodiesterase from bovine brain interchangeably and both proteins displaced ¹²⁵I-calmodulin from high-affinity binding sites on human erythrocyte ghosts with equal effectiveness. This procedure yielded a labeling stoichiometry of 1.34. Scatchard plots of binding of ¹²⁵I-calmodulin to ghosts were consistent with the presence of a single class of high-affinity binding sites with the properties expected of (Ca²⁺ + Mg²⁺)-ATPase molecules. The binding showed positive cooperativity and occurred only in the presence of Ca²⁺. The maximum amount of binding seen in Scatchard plots corresponded to 4.1 × 10³ sites per ghost.     

Calmodulin binding to platelet plasma membranes

Calmodulin copurifies with platelet plasma membranes isolated by glycerol-induced lysis and density gradient centrifugation. These membranes also bind ¹²⁵I-labeled calmodulin in vitro in the presence of Ca²⁺. Binding is largely reduced by replacing Ca²⁺ by Mg²⁺ or by addition of an excess unlabeled calmodulin. The specific component of binding is saturable, with an apparent K_d of 27 nM and a maximum of 15.9 pmol binding sites per mg of membrane protein. This is equivalent to approx. 4100 binding sites per platelet. Binding was inhibited by addition of phenothiazines, a group of calmodulin antagonists. Half-maximal inhibition was attained with approx. 20 μM trifluoperazine or 50 μM chlorpromazine. In contrast, chlorpromazine-sulfoxide which is inactive towards calmodulin, did not affect the binding. Calmodulin binding polypeptides of the plasma membrane were identified by a gel-overlay technique. A major calmodulin-binding component of molecular weight 149 000 was detected. Binding to this band was Ca²⁺-dependent and inhibited by chlorpromazine. The molecular weight of this polypeptide is similar to that of glycoprotein I and also that of the red cell (Ca²⁺ + Mg²⁺)-stimulated ATPase, which is known to bind calmodulin. The possible role of calmodulin in platelet activation is analysed.