Two classes of arginine vasopressin binding sites on rat brain membranes

Specific binding sites for arginine vasopressin (AVP) were demonstrated on rat brain membranes using [³H]AVP of high specific activity. At pH 7.4 in the presence of 5 mM MgCl₂, one class of sites was measured with aK _D of 0.56 nM and aB _max of 4.3 fmol/mg protein. At pH 8.0 in the presence of MgCl₂, two distinct sites were observed, havingK _D values of 0.42 and 13 nM andB _max values of 5.6 and 68 fmol/mg protein, respectively, and similar results were obtained at pH 7.4 after repeatedly freezing and thawing the membranes. Binding increased with pH, apparently representing increased occupancy of the high capacity, lower affinity site. Binding to the lower affinity site was also enhanced by freezing and thawing membranes, or by adding 5 mM NiCl₂ or 10 M ZnCl₂ to the incubation medium, whereas binding to the high affinity site was dependent on the addition of Mg. AVP was over 35 times more active in displacing 0.4 nM AVP than oxytocin or arginine-vasotocin, and 10,000 times more active than somatostatin. A number of other peptides had no effect on [³H]AVP binding at concentrations up to 10^–5 M. Autoradiography and regional dissection studies revealed a marked concentration of high affinity AVP-binding sites in the supraoptic and paraventricular nuclei of the hypothalamus, and Mg significantly enhanced the binding in these regions.     

Purification and Characterization of Aspartic Protease Derived from Sf9 Insect Cells

An aspartic protease that is significantly produced by baculovirus-infected Spodoptera frugiperda Sf9 insect cells was purified to homogeneity from a growth medium. To monitor aspartic protease activity, an internally quenched fluoresce (IQF) substrate specific to cathepsin D was used. The purified aspartic protease showed a single protein band on SDS–PAGE with an apparent molecular mass of 40 kDa. The N-terminal amino acid sequence of the enzyme had a high homology to a Bombyx mori aspartic protease. The enzyme showed greatest affinity for the IQF substrate at pH 3.0 with a K _m of 0.85 μM. The k _cat and k _cat?K _m values were 13 s^?1 and 15 s^?1 μM^?1 respectively. Pepstatin A proved to be a potent competitive inhibitor with inhibitor constant, K _i, of 25 pM.     

The pre-steady state reaction of ferrocytochrome c with the cytochrome c-cytochrome aa3 complex

1. Using stopped-flow technique we have investigated the electron transfer form cytochrome c to cytochrome aa₃ and to the (porphyrin) cytochrome c-cytochromeaa₃ complex.2. In a low ionic strength medium, the pre-steady state reaction occurs in a biphasic way with rate constants of at least 2 · 10⁸ M^?1 · s^?1 and about 10⁷ M^?1 · s^?1 (I = 8.8 mM, pH 7.0, 10° C), respectively.3. A comparison of the rate constants, determined in the presence of an excess of cytochrome c with those found in the presence of an excess of cytochrome aa₃ reveals the existence of two slower reacting sites on the functional unit (2 hemes and 2 coppers) of cytochrome aa₃. On basis of these results we discuss various models. If no site-site interactions are assumed (non-cooperative model) cytochrome aa₃ has 2 high and 2 low affinity sites available for the reaction with ferrocytochrome c. If negative cooperativity occurs, cytochrome aa₃ has 2 high affinity sites which change into 2 low affinity sites upon binding of one cytochrome c molecule. The latter model is favoured.     

Characterization and Subcellular Localization of l-[3H]Carnitine Binding Sites in Rat Brain

Abstract: In the present study, we investigated the existence of a binding site for l -carnitine in the rat brain. In crude synaptic membranes, l -[³H]carnitine bound with relatively high affinity (K_D = 281 nM) and in a saturable manner to a finite number (apparent B_max value = 7.3 pmol/mg of protein) of binding sites. Binding was reversible and dependent on protein concentration, pH, ionic strength, and temperature. Kinetic studies revealed a K_off of 0.018 min^?1 and a K_on of 0.187 × 10^?3 min^?1 nM^?1. Binding was highest in spinal cord, followed by medulla oblongata-pons ≥ corpus striatum ≥ cerebellum = cerebral cortex = hippocampus = hypothalamus = olfactory bulb. l -[³H]Carnitine binding was stereoselective for the l -isomers of carnitine, propionylcarnitine, and acetylcarnitine. The most potent inhibitor of l -[³H]carnitine binding was l -carnitine followed by propionyl-l -carnitine. Acetyl-l -carnitine and isobutyryl-l -carnitine showed an affinity ～500-fold lower than that obtained for l -carnitine. The precursor γ-butyrobetaine had negligible activity at 0.1 mM. l -Carnitine binding to rat crude synaptic membrane preparation was not inhibited by neurotransmitters (GABA, glycine, glutamate, aspartate, acetylcholine, dopamine, norepinephrine, epinephrine, 5-hydroxytryptamine, histamine) at a final concentration of 0.1 mM. In addition, the binding of these neuroactive compounds to their receptors was not influenced by the presence of 0.1 mMl -carnitine. Finally, a subcellular fractionation study showed that synaptic vesicles contained the highest density of l -carnitine membrane binding sites whereas l -carnitine palmitoyltransferase activity was undetectable, thus excluding the possibility of the presence of an active site for carnitine palmitoyltransferase. This finding indicated that the localization of the l -[³H]carnitine binding site should be essentially presynaptic.     

Characterization and properties of steroid binding protein in Bufo arenarum serum

Serum steroid binding properties of mature Bufo arenarum females were studied. Binding data obtained using charcoal adsorption assay and equilibrium dialysis methods indicates a single protein, named Bufo arenarum sex binding protein (Ba SBP), which binds 5 α-dihydrotestosterone (DHT), testosterone (T), and estradiol-17β (E₂) with high affinity (10⁷ M^?1 – 10⁸ M^?1) and fair capacity (10^?6 M). Scatchard plot analysis demonstrated the coexistence of two binding sites. Ba SBP has a sedimentation coefficient of 5.2 S in sucrose gradient centrifugation in low salt and under steady-state conditions. The specificity of this protein, determined by competitive binding experiments, is comparable to human SBP. DHT and T bind with higher affinity than E₂. Estriol and estrone competed poorly, while diethylstilbestrol and C₂₁ steroids did not compete. The binding capacity of this protein is under estrogenic control. © 1994 Wiley-Liss, Inc.     

The low affinity binding sites of the vitamin D-dependent calcium-binding protein and their functional role in calcium transport

Calcium- and other divalent cation-binding properties of the vitamin D-dependent calcium-binding protein isolated from the duodenal mucosa of chicks were studied using the flow dialysis technique and ⁴⁵Ca. It was found that the calcium-binding protein along with the high affinity binding sites has approximately 40 low affinity binding sites with K_a of about 1000 M^?1. The low affinity sites possess of certain specificity towards binding of Ca. The affinity of the calcium-bindin protein for other divalent cations depends on the ionic radius. It is suggested that the low affinity binding sites of the calcium-binding protein take part in calcium transport organization across the intestinal epithelium.     

Two modes of bicarbonate utilization in the marine green macroalga Ulva lactuca

The green marine macroalga Ulva lactuca L. was found to be able to utilize HCO₃^? from sea water in two ways. When grown in flowing natural sea water at 16°C under constant dim irradiance, photosynthesis at pH8.4 was suppressed by acetazolamide but unaffected by 4,4′-diisothiocyanostilbene-2,2′-disulphonate. These responses indicate that photosynthetic HCO₃^? utilization was via extracellular carbonic anhydrase (CA) -mediated dehydration followed by CO₂ uptake. The algae were therefore described as being in a ‘CA state’. If treated for more than 10 h in a sea water flow-through system at pH9.8, these thalli became insensitive to acetazolamide but sensitive to 4,4′-diisothiocyanostilbene-2,2′-disulphonate. This suggests the involvement of an anion exchanger (AE) in the direct uptake of HCO₃^?, and these plants were accordingly described as being in an ‘AE state’. Such thalli showed an approximately 10-fold higher apparent affinity for HCO₃^? (at pH9.4) than those in the ‘CA state’, while thalli of both states showed a very high apparent affinity for CO₂. These results suggest that the two modes of HCO₃^? utilization constitute two ways in which inorganic carbon may enter the Ulva lactuca cells, with the direct entry of HCO₃^?, characterizing the ‘AE state’, being inducible and possibly functioning as a complementary uptake system at high external pH values (e.g. under conditions conducive to high photosynthetic rates). Both mechanisms of entry appear to be connected to concentrating CO₂ inside the cell, probably via a separate mechanism operating intracellularly.     

Specific somatostatin-binding to cytosol of bovine gallbladder mucosa

The binding of somatostatin was studied in the cytosolic fraction of bovine gallbladder mucosa. The binding reaction depended on time, temperature and pH, and was reversible, saturable and specific. Stoichiometric data suggested the presence of two classes of binding sites: a class with high affinity (K _d=23.6 nM) and low capacity (3.7 pmol somatostatin/mg protein) and a class with low affinity (K _d=284.6 nM) and high capacity (85.0 pmol somatostatin/mg protein) at 37°C and pH 7.4. The binding sites were highly specific for somatostatin since peptides such as [Leu]enkephalin, neurotensin, vasoactive intestinal peptide and substance P showed practically no effect upon somatostatin binding. The presence of somatostatin-binding sites in the cytosolic fraction of gallbladder mucosa, together with the known occurrence of somatostatin nerve endings in the gallbladder strongly suggests that this peptide may be involved in the physiology and physiopathology of gallbladder mucosa.     

Interaction of vinblastine with steady-state microtubules in vitro

At low concentrations, vinblastine binds rapidly and reversibly to a very limited number of high affinity sites on steady-state bovine brain microtubules (mean K_d, 1.9 × 10^?6m; 16.8 ± 4.3 vinblastine binding sites per microtubule) which appear to be located at one or both ends of the microtubules. At high concentrations, vinblastine binds to a high binding capacity class of sites of undetermined affinity, located on helical strands of protofilaments which form at the ends of depolymerizing microtubules, and/or along the surface of the microtubules. Substoichiometric inhibition of microtubule assembly, which occurs at low vinblastine concentrations, appears to be due to the binding of vinblastine to the high affinity class of sites. Fifty per cent inhibition of tubulin addition to the net assembly ends of steady-state microtubules occurred at 1.38 × 10^?7m-drug, and at this concentration, 1.16 ± 0.27 molecules of vinblastine were bound to the high affinity class of sites. Vinblastine appeared to bind directly to the microtubule ends, and our results indicate that vinblastine inhibits the assembly of steady-state bovine brain microtubules by binding rapidly and with high affinity to one or two molecules of tubulin at the net assembly ends. Splaying and peeling of protofilaments at microtubule ends and the active depolymerization of microtubules occurred only at vinblastine concentrations greater than 1 × 10^?6 to 2 × 10^?6m. This action of vinblastine is associated with and may be due to the binding of vinblastine to the high capacity class of sites. Both actions of vinblastine may be due to the binding of vinblastine to the same binding sites on the tubulin molecule, with the sites exhibiting either a high or low affinity depending upon the location in the microtubule.     

PHOTOSYNTHETIC PROPERTIES OF PROTOPLASTS,AS COMPARED WITH THALLI,OF ULVA FASCIATA (CHLOROPHYTA)1

Protoplasts were prepared from Ulva fasciata Delile, and their photosynthetic performance was measured and compared with that of thalli discs. These protoplasts maintained maximal rates of photosynthesis as high as those of thalli (up to 300 μmol O₂·mg chlorophyll^?1·h^?1) for several hours after preparation and were therefore considered suitable for kinetic studies of inorganic carbon utilization. The photosynthetic K_1/2(inorganic carbon) at pH 6.1 was 3.8 μM and increased to 67, 158, and 1410 μM at the pH values 7.0, 7.9, and 8.9, respectively. Compared with these protoplasts, thalli had a much lower affinity for CO₂ but approximately the same affinity for HCO₃^?. Comparisons between rates of photosynthesis and the spontaneous dehydration of HCO₃^? (at 50 μM inorganic carbon) revealed that photosynthesis of both protoplasts (which lacked apparent activity of extracellular/surface-bound carbonic anhydrase) and thalli (which were only 25% inhibited by the external carbonic anhydrase inhibitor acetazolamide) could not be supported by CO₂ formation in the medium at the higher pH values, indicating HCO₃^? uptake. Since both protoplasts and thalli were sensitive to 4,4′-diisothiocyanostilbene-2,2′-disulfonate, we suggest that HCO₃^? transport was facilitated by the membrane-located anion exchange protein recently reported to function in certain Ulva thalli. These findings suggest that the presence of a cell wall may constitute a diffusion barrier for CO₂, but not for HCO₃^?, utilization under natural seawater conditions.     

Specific binding sites for d-α-tocopherol on human erythrocytes

Since vitamin E deficiency is associated with increased susceptibility of erythrocytes to hemolysis, we investigated the presence of tocopherol binding sites in human red blood cells. Erythrocytes were found to have specific binding sites for d-α-[³H]tocopherol with properties of receptors. Kinetic studies of binding demonstrated two binding sites: one with high affinity (equilibrium association constant K_a = 2.6·10⁷ M^?1), low capacity (7600 sites/cell) and the second with low affinity (K_a = 1.24·10⁶ M^?1), high capacity (150000 sites/cell). These sites are at least partly protein in nature.     

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.     

High and low affinity binding sites for Concanavalin A on normal human fibroblasts in vitro

The binding of high specific activity, radioactive Concanavalin A to cultured normal human fibroblasts was investigated. We report the presence of two classes of Concanavalin A binding sites on the plasma membranes of these cells. These classes of binding sites are distinguished by their affinities for the lectin. Scatchard analysis of the binding data indicates the presence of a class of high affinity sites which are saturated at about 0.25 μg/ml of Concanavalin A. The other, lower affinity binding sites are not saturated until 50–100 μg/ml Concanavalin A levels are achieved. At 4°C the K_a for the high affinity sites varies between 1.5 – 5 × 10⁹ M^?1 depending on the method used to label the Concanavalin A. For the lower affinity sites K_a varies between 1 – 4 × 10⁶ M^?1. The average number of high affinity sites per cell is 8 × 10⁵ representing less than 1% of the total receptor sites for the lectin.     

Inorganic carbon transport across cell compartments of the halotolerant alga Dunaliella salina

The inorganic carbon (C_i) accumulation and the intracellular location of carbonic anhydrase (CA, EC 4.2.1.1) in the halotolerant unicellular alga Dunaliella salina have been investigated. The rate of HCO₃ -dependent O₂ evolution was determined by growth conditions. Algae grown under high CO₂ conditions (5% CO₂ in air, v/v; high C_i cells) had a very low affinity for HCO₃^? at pH 7.0 and 8.2, whereas algae grown under low CO₂ conditions (0.03% CO₂ in air; low C_i cells) showed a high affinity for HCO₃^? at both pH values and were sensitive to Dextran-bound sulfonamide (DBS), an inhibitor of extracellular CA. The photosynthetic rate or HCO₄^? dependent O₂ evolution was always higher at pH 7.0 than at pH 8.2. Ethoxyzolamide (EZ), an inhibitor of total (extacellular plus intracellular) CA activity, strongly inhibited photosynthesis at both pH values. During adaptation from high to low CO₂ conditions CA activity increased in chloroplasts in a process dependent on the novo protein synthesis. Carbonic anhydrase activity was found in the supernatant and pellet fractions of chloroplast homogenates. The rate of photosynthesis of chloroplasts from low C_i cells was higher at pH 7.0 than at pH 8.2. The alkalinization of the growth medium, which took place only in the presence of C_i, was partially inhibited by DBS and completely by EZ. We suggest that in D. salina CO₂ is the general form of C_i transported across the plasma membrane and the chloroplast envelope and that bicarbonate enters the cell mainly, although not entirely, by an ‘indirect’ mechanism after dehydration to CO₂.     

Characterisation of the Binding of [3H]WAY-100635, a Novel 5-Hydroxytryptamine1A Receptor Antagonist, to Rat Brain

Abstract: The specific binding of [³H]WAY-100635 {N-[2-[4-(2-[O-methyl-³H]methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide trihydrochloride} to rat hippocampal membrane preparations was time, temperature, and tissue concentration dependent. The rates of [³H]WAY-100635 association (k₊₁ = 0.069 ± 0.015 nM^?1 min^?1) and dissociation (k_?1 = 0.023 ± 0.001 min^?1) followed monoexponential kinetics. Saturation binding isotherms of [³H]WAY-100635 exhibited a single class of recognition site with an affinity of 0.37 ± 0.051 nM and a maximal binding capacity (B_max) of 312 ± 12 fmol/mg of protein. The maximal number of binding sites labelled by [³H]WAY-100635 was ～36% higher compared with that of 8-hydroxy-2-(di-n-[³H]-propylamino)tetralin ([³H]8-OH-DPAT). The binding affinity of [³H]WAY-100635 was significantly lowered by the divalent cations CaCl₂ (2.5-fold; p < 0.02) and MnCl₂ (3.6-fold; p < 0.05), with no effect on B_max. Guanyl nucleotides failed to influence the K_D and B_max parameters of [³H]WAY-100635 binding to 5-HT_1A receptors. The pharmacological binding profile of [³H]WAY-100635 was closely correlated with that of [³H]8-OH-DPAT, which is consistent with the labelling of 5-hydroxytryptamine_1A (5-HT_1A) sites in rat hippocampus. [³H]WAY-100635 competition curves with 5-HT_1A agonists and partial agonists were best resolved into high- and low-affinity binding components, whereas antagonists were best described by a one-site binding model. In the presence of 50 µM guanosine 5′-O-(3-thiotriphosphate) (GTPγS), competition curves for the antagonists remained unaltered, whereas the agonist and partial agonist curves were shifted to the right, reflecting an influence of G protein coupling on agonist versus antagonist binding to the 5-HT_1A receptor. However, a residual (16 ± 2%) high-affinity agonist binding component was still apparent in the presence of GTPγS, indicating the existence of GTP-insensitive sites.     

A multispecific monoclonal antibody G2 recognizes at least three completely different epitope sequences with high affinity

A monoclonal antibody (mAb) G2 possesses an unusual characteristic of reacting with at least three proteins (ATP6V1C1, SEPT3, and C6H10orf76) other than its original antigen, chicken prion protein (ChPrP). The epitopes on ChPrP and ATP6V1C1 have been identified previously. In this study, we identified the epitope in the third protein, SEPT3. Interestingly, there was no amino acid sequence similarity among the epitopes on the three proteins. These epitopes had high binding affinities to G2 (K _D = ～10^?7 M for monovalent binding and K _D = ～10^?9 M for divalent binding), as determined using a SPR biosensor. This is the first report on a three‐in‐one mAb recognizing completely different epitope sequences with high affinity. Additionally, competitive ELISA indicated that the binding sites on G2, specific for the three different epitopes, overlapped, suggesting that the antigen‐binding site may be flexible in the free form and capable of adapting to at least three different conformations to enable interactions with three different antigens.     

Binding of β1-adrenoreceptor antagonist [3H]CGP 26505 in rat cerebral cortex and sinus atrial nodeantagonist [3H]CGP 26505 in rat cerebral cortex and sinus atrial node

Specific β₁-adrenoreceptors antagonist [³H]CGP 26505 binding was characterized in rat cerebral cortex and heart sinus atrial node. In both tissues [³H]CGP 26505 binding was maximal at 25°C, it was specific, saturable and protein concentration dependent. Scatchard analysis of saturation isotherms of specific [³H]CGP 26505 binding in cerebral cortex showed that [³H]CGP 26505 binds a single class of high affinity sites with a dissociation constant (K_D) of 1±0.3 nM and a maximal number of binding sites (B_max) of 40±2 fmol/mg of protein. In sinus atrial node, [³H]-CGP 26505 binds a single class of high affinity sites (K_D=1.9±0.4 nM, B_max=28±2 fmol/mg of protein).     

Kinetic Properties of Fungal Polyamine Oxidases and Their Application to Differential Determination of Spermine and Spermidine

Polyamine oxidase from Penicillium chrysogenum oxidized spermine rapidly and spermidine slightly at pH 7.5. The apparent Km values for spermine and spermidine were calculated to be 2.25 × 10^?5 m and 9.54 × 10^?6 m, respectively. The relative maximum velocities for spermine and spermidine were 3.37 × 10^?3 m (H₂O₂) per min per mg of protein and 2.08 × 10^?4 m (H₂O₂) per min per mg of protein, respectively. Spermine oxidation of the enzyme was competitively inhibited by spermidine and putrescine. The apparent Ki values by spermidine and putrescine were calculated to be 3.00 × 10^?5 m and 1.80 × 10^?8 m, respectively. On the other hand, polyamine oxidase from Aspergillus terreus rapidly oxidized both spermidine and spermine at pH 6.5. The apparent Km values for spermidine and spermine were 1.20 × 10^?8 m and 5.37 × 10^?7 m, respectively. The relative maximum velocities for spermidine and spermine were 1.55 × 10^?2 m (H₂O₂) per min per mg of protein and 6.20 × 10^?3 m (H₂O₂) per min per mg of protein, respectively.

Differential determination of spermine and spermidine was carried out using the two enzymes. The initial rate was assayed with Penicillium enzyme and the end point was measured afte addition of Aspergillus enzyme. Small amounts of polyamines (25 to 200 nmol of spermine and 25 to 250 nmol of spermidine) were assayed by solving two simultaneous equations obtained from the rate assay method and the end point assay method. The calculated values were in close agreement with those obtained by an amino-acid analyzer.     

Ecdysteroid receptors in a tumorous blood cell line of Drosophila melanogaster

The tumorous Drosophila melanogaster blood cell line BII has been studied for evidence for the presence of ecdysteroid receptors. The [³H]ponasterone A (pon A)* used in this study has been extensively purified, and the location of the tritium in the molecule has been partially determined. BII cells do not metabolise ecdysteroids. Intact cells demonstrate a considerable specific uptake of [³H]pon A which is saturable, apparently showing two specific components: a very high affinity component (K_D = 0.3 nM) and a high affinity component (K_D = 2 nM). The specific binding of [³H]pon A to whole cells is compatible with unlabelled ecdysteroids, but not with mammalian steroid hormones. The association rate constant (k_a) for [³H]pon A was determined to be 3 × 10⁷M^?1min^?1 at 21 °C, while the dissociation rate constant (k_d) for the specifically bound [³H]pon A was found to be 4.4 × 10^?3/min. Together, the kinetic rate constants yield a value of 0.15 nM for the K_D. The receptors have been partially characterised in a cell-free extract prepared by sonification of the cells. The optimum pH for extraction and hormone binding is 8.2. Scatchard plots of binding data indicate that the cell-free extract also contains two high affinity specific binding components (k_D = 0.1 nM and K_D = 1 nM). The hgih affinity binders are macromolecular, as shown by chromatography on Sephadex G-25, and are susceptible to protease digestion, heat, and treatment with N-ethylmaleimide. Sucrose density centrifugation of the labelled receptor shows one peak at approximately 6S. The stability of the receptor preparation has been studied and conditions have been empirically determined (10% w/v sucrose, 25 mM dithioerthreitol, and 10 mM citrate), whereby the binding capacity of the unlabelled receptor is stable for at least 8 weeks if frozen at ?20°C.