Inhibition of atrial natriuretic peptide-induced cyclic GMP accumulation in the bovine endothelial cells with anti-atrial natriuretic peptide receptor antiserum

Using an antiserum raised against the purified atrial natriuretic peptide (ANP) receptor that has a disulfide-linked homodimeric structure and represents one subtype of the multiple ANP receptors, we showed that the receptor is coupled to the guanylate cyclase activation; formerly, this type of ANP receptor is not considered to be coupled to the cyclase. The specificity of the antiserum was determined by immunoblot analysis and immunoprecipitation. The anti-receptor antiserum did not compete with 125I-ANP for binding to the receptor but it lowered the affinity of the receptor. When added to bovine endothelial cell cultures, the antiserum blocked the cyclic GMP response of the cells triggered by ANP. These results indicate that the subtype of the ANP receptor recognized by the antiserum is responsible for the activation of particulate guanylate cyclase as well as the double function type receptor that has been assumed to contain both the receptor domain and the catalytic domain for cGMP synthesis on the same molecule. The presence of dissociative complexes of ANP receptor and particulate guanylate cyclase was also demonstrated by radiation inactivation analysis.     

Expression,purification, and characterization of the intra-cellular domain of the ANP receptor

The membrane-bound atrial natriuretic peptide receptor (GCA) catalyzes the formation of cGMP from GTP in response to natriuretic peptide hormones. Previous structural studies have focused on the extra-cellular hormone binding domain of this receptor whereas its intra-cellular domain has not yet been amenable to such studies. We report here the baculovirus expression and purification of the GCA intra-cellular domain construct GCA_ID comprising the complete intra-cellular region which includes the kinase-homology domain, coiled-coil region, and catalytic cyclase domain. The intra-cellular domain was enzymatically characterized in terms of guanylyl cyclase activity and the effects of ATP, manganese, and Triton X-100. Our results indicate that the activity of the intra-cellular domain of the ANP receptor is about 2 fold less active compared to a truncated cyclase domain construct lacking the kinase-like domain that was also expressed and purified. In addition, unlike the full length receptor, the intra-cellular domain could not be activated by Triton X-100/Mn²⁺ or its activity stimulated by ATP. These data therefore indicate that the major part of the transition from the basal state to the fully, ANP/ATP-dependent, activated state as well its stimulation/enhancement by Triton X-100/Mn²⁺ requires the full length receptor. These receptor insights could aid in the development of novel therapeutics as the GCA receptor is a key drug target for cardiovascular diseases.     

Proposed mechanisms of stimulation and inhibition of guanylate cyclase with reference to the actions of chlorpromazine,phospholipases and triton X-100

Particulate guanylate cyclase activity in the homogenate of guinea pig tracheal muscle was activated prominently after treatment with phospholipase-A or C. Even after the stimulation by phospholipases or Triton X-100, most of the activity was still associated with the particulate fraction. Phenothiazine tranquilizers and imipramine strongly inhibited the activity, whereas haloperidol did not. These results suggest that (i) particulate guanylate cyclase is associated fimly with the membrane and perturbation of the membrane architecture rather than solubilization of the enzyme is accountable for the stimulation, and (ii) stabilization of membranes in such a way that it affects the function of mucarinic cholinergic receptors inhibits the activity.     

Physical and functional association of the atrial natriuretic peptide receptor with particulate guanylate cyclase as demonstrated using detergent extracts of bovine lung membranes

Coupling of the atrial natriuretic peptide (ANP) receptor to particulate guanylate cyclase has been demonstrated kinetically and chromatographically using bovine lung plasma membranes and their detergent extracts. Addition of ANP to the membrane suspension stimulated guanylate cyclase activity 2-5-fold indicating the presence of ANP-sensitive particulate guanylate cyclase. The enzyme retained the ability to respond to ANP even after solubilization with digitonin. Characterization of the solubilized enzyme by gel filtration and affinity chromatography revealed that the ANP receptor and particulate guanylate cyclase exist as a functionally but not covalently linked stable complex.     

The guanylate cyclase/receptor family of proteins

Guanylate cyclase, which catalyzes the formation of cGMP from GTP, exists in both the soluble and particulate fractions of cells. At least two different cellular compartments for the particulate enzyme exist: the plasma membrane and cytoskeleton. The enzyme form found in the soluble fraction is a heterodimer that can be regulated by free radicals and nitrovasodilators, whereas the membrane form exists as a single-chain polypeptide that can be regulated by various peptides. These peptides include resact and speract obtained from eggs and atrial natriuretic peptides (ANP). The species of guanylate cyclase present in cytoskeletal fractions resists solubilization with non-ionic detergents; its structural properties are not yet known. cDNAs encoding the membrane form of guanylate cyclase have been isolated from different tissues and species, and in all cases the DNA sequences predict a protein containing a single transmembrane domain. The carboxyl (intracellular) domain is highly conserved from sea urchins through mammals, whereas the extracellular domain (amino terminus) varies considerably. The predicted amino acid sequences demonstrate that the membrane form of guanylate cyclase is a member of a diverse and complex family of proteins that includes a low molecular weight ANP receptor, protein kinases, and the cytoplasmic form of guanylate cyclase. cDNA encoding a membrane form of the enzyme from mammalian tissues has been expressed in cultured cells, and the expressed guanylate cyclase specifically binds ANP and is activated by ANP. The membrane form of guanylate cyclase, then, serves as a cell surface receptor, representing the first recognized protein to directly catalyze formation of a low molecular weight second messenger in response to ligand binding.     

Human atrial natriuretic peptide receptor defines a new paradigm for second messenger signal transduction.

We isolated cDNAs encoding a 115 kd human atrial natriuretic peptide (alpha ANP) receptor (ANP-A receptor) that possesses guanylate cyclase activity, by low-stringency hybridization with sea urchin Arbacia punctulata membrane guanylate cyclase probes. The human ANP-A receptor has a 32 residue signal sequence followed by a 441 residue extracellular domain homologous to the 60 kd ANP-C receptor. A 21 residue transmembrane domain precedes a 568 residue cytoplasmic domain with homology to the protein kinase family and to a subunit of the soluble guanylate cyclase. COS-7 cells transfected with an ANP-A receptor expression vector displayed specific [125I]alpha ANP binding, and exhibited alpha ANP stimulated cGMP production. These data demonstrate a new paradigm of cellular signal transduction where extracellular ligand binding allosterically regulates cyclic nucleotide second-messenger production by a receptor cytoplasmic catalytic domain.     

Particulate guanylate cyclase of skeletal muscle: effects of Ca2+ and other divalent cations on enzyme activity.

The properties of particulate guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) from purified rabbit skeletal muscle membrane fragments were studied. Four membrane fractions were prepared by sucrose gradient centrifugation and the fractions characterized by analysis of marker enzymes. Guanylate cyclase activity was highest in the fraction possessing enzymatic properties typical of sarcolemma, while fractions enriched with sarcoplasmic reticulum had lower activities. In the presence of suboptimal Mn2+ concentrations, Mg2+ stimulated particulate guanylate cyclase activity both before and after solubilization in 1% Triton X-100. Guanylate cyclase activity was biphasic in the presence of Ca2+. Increasing the Ca2+ concentration from 10(-8) to 10(-5) M decreased the specific activity. As the Ca2+ concentration was further increased to 5 . 10(-4) M enzyme activity again increased. After solubilization of the membranes in 1% Triton X-100, Ca2+ suppressed enzyme activity. Studies utilizing ionophore X537A indicated that the altered effect of Ca2+ upon the solubilized membranes was independent of asymmetric distribution of Ca2+ and Mg2+.     

Manganese and magnesium dependent properties and inner plasma membrane surface localization of guanylate cyclase from murine plasmocytoma cells

The particulate fraction from murine plasmocytoma cells contained 90 per cent of the total guanylate cyclase activity. Triton X-100 produced a 6 fold stimulation of guanylate cyclase activity in plasma membrane enriched fractions obtained by zonal centrifugation. Isolated inside out (10) vesicles contained 9 times more activity than rightside out (RSO) vesicles. This difference was abolished by Triton X-100 treatment of the vesicles indicating that the catalytic site of guanylate cyclase is located on the inner face of the plasma membrane. Kinetic studies of membranous guanylate cyclase showed that optimal activity was found with manganese. Only 20 per cent of this activity was obtained with magnesium. The Km for GTP with magnesium (1.4 mM) was about 7 fold greater than with manganese (0.2 mM). Positive cooperativity was obtained in both cases and the Hill coefficients were 1.8 for manganese and 1.6 for magnesium. Physiological concentrations of ATP were found to inhibit both manganese and magnesium supported activities indicating a possible regulatory mechanism for this nucleotide in vivo.     

Mechanism of activation of particulate guanylate cyclase by atrial natriuretic peptide as deduced from radiation inactivation analysis

The interaction between the receptor (Rc) for atrial natriuretic peptide (ANP) and the effector enzyme particulate guanylate cyclase (GC) has been studied by radiation inactivation. Irradiation of bovine lung membranes produced an increase in GC activity at low radiation doses followed by a dose-dependent reduction at higher doses. This deviation from linearity in the inactivation curve disappeared when lung membranes were pretreated with ANP. Essentially identical results were also obtained with adrenal membranes. Based on these radiation inactivation data, the following dissociative mechanism of activation of particulate guanylate cyclase by ANP has been proposed: Rc.GC(inactive) + ANP----Rc.ANP + GC(active).     

Atrial natriuretic peptide binding, cross-linking, and stimulation of cyclic GMP accumulation and particulate guanylate cyclase activity in cultured cells

The stimulation of cyclic GMP accumulation and particulate guanylate cyclase activity by atrial natriuretic peptide (ANP) was compared to the affinity and number of ANP receptors in eight cultured cell types. At 100 nM, ANP increased cyclic GMP by 13-fold in bovine adrenal cortical, 35-fold in human lung fibroblast, 58-fold in canine kidney epithelial, 60-fold in bovine aortic smooth muscle, 120-fold in rat mammary epithelial, 260-fold in rat Leydig, 300-fold in bovine kidney epithelial, and 475-fold in bovine aortic endothelial cells. ANP (1 microM) increased particulate guanylate cyclase activity by 1.5-, 2.5-, 3.1-, 3.2-, 5.0-, 7.0-, 7.8-, and 8.0-fold in bovine adrenal cortical, bovine aortic smooth muscle, human lung fibroblast, canine kidney epithelial, rat mammary epithelial, rat Leydig, bovine kidney epithelial, and bovine aortic endothelial cells, respectively. Specific 125I-ANP binding to intact rat Leydig (3,000 sites/cell; Kd = 0.11 nM), bovine aortic endothelial (14,000 sites/cell; Kd = 0.09 nM), bovine adrenal cortical (50,000 sites/cell; Kd = 0.12 nM), human lung fibroblast (80,000 sites/cell; Kd = 0.32 nM), and bovine aortic smooth muscle (310,000 sites/cell; Kd = 0.82 nM) cells was saturable and high affinity. No specific and saturable ANP binding was detected in bovine and canine kidney epithelial and rat mammary epithelial cells. Two ANP-binding sites of 66,000 and 130,000 daltons were specifically labeled by 125I-ANP after cross-linking with disuccinimidyl suberate. The 130,000-dalton ANP-binding sites bound to a GTP-agarose affinity column, and the specific activity of guanylate cyclase was increased by 90-fold in this fraction. Our results demonstrate that the increase in cyclic GMP accumulation and particulate guanylate cyclase activity by ANP does not correlate with the affinity and number of ANP-binding sites. These results suggest that multiple populations of ANP receptors exist in these cells and that only one receptor subtype (130,000 daltons) is associated with particulate guanylate cyclase activity.     

Ligand binding-dependent limited proteolysis of the atrial natriuretic peptide receptor: juxtamembrane hinge structure essential for transmembrane signal transduction

The atrial natriuretic peptide (ANP) receptor is a 130-kDa transmembrane protein containing an extracellular ANP-binding domain, a single transmembrane sequence, an intracellular kinase-homologous domain, and a guanylate cyclase (GCase) domain. We observed that the receptor, when bound with ANP, was rapidly cleaved by endogenous or exogenously added protease to yield a 65-kDa ANP-binding fragment. No cleavage occurred without bound ANP. This ligand-induced cleavage abolished GCase activation by ANP. Cleavage occurred in an extracellular, juxtamembrane region containing six closely spaced Pro residues and a disulfide bond. Such structural features are shared among the A-type and B-type ANP receptors but not by ANP clearance receptors. The potential role of the hinge structure was examined by mutagenesis experiments. Mutation of Pro(417), but not other Pro residues, to Ala abolished GCase activation by ANP. Elimination of the disulfide bond by Cys to Ser mutations yielded a constitutively active receptor. Pro(417), and Cys(423) and Cys(432) forming the disulfide bond are strictly conserved among GCase-coupled receptors, while other residues are largely variable. The conserved Pro(417) and the disulfide bond may represent a consensus signaling motif in the juxtamembrane hinge structure that undergoes a marked conformational change upon ligand binding and apparently mediates transmembrane signal transduction.     

Natriuretic peptide receptor: Structure and signaling

The ANP receptor is a single-transmembrane sequence receptor coupled to guanylate cyclase (GCase). It belongs to a family of GCase-coupled receptors that share a common overall molecular configuration. Collectively, theses GCase-coupled receptors belong to a larger family of single-transmembrane sequence receptors that include growth hormone and cytokine receptors. The signal transduction mechanism of these receptors has not been thoroughly understood. Receptor dimerization (or oligomerization) has been suggested as the mechanism. However, at least for the ANP receptor, dimerization has been seen to occur in the absence of the ligand, suggesting that an additional, as yet unknown effect of hormone binding is responsible for receptor activation. To understand the signaling mechanism, some of the functions and subsites of the ANP receptor critical for signaling have been identified, including the binding stoichiometry, receptor self-association, the juxtamembrane hinge structure containing a signature motif critical for GCase signaling, ANP-binding site residues, chloride-dependence of ANP binding, disulfide linkages, and glycosylation structures. These structures and the functional sites have been identified in the crystal structure of dimerized recombinant extracellular domain of the ANP receptor. The intracellular domain contains a kinase-homologous domain that regulates the activity of the GCase domain responding to ANP binding and also to binding of the allosteric effector ATP. Moreover, this regulatory role of the kinase-homologous domain is modulated by its own phosphorylated state. Although considerable data have been accumulated, the mechanism of ANP receptor signaling has not been well defined. Further studies are necessary to understand how ANP binds to the receptor, what conformational effect is caused by ANP binding, how this effect is transduced across the cell membrane, and how this transmembrane effect leads to stimulation of the GCase catalytic activity.     

ANF stimulation of detergent-dispersed particulate guanylate cyclase from bovine adrenal cortex

Particulate guanylate cyclase from bovine adrenal cortex can be stimulated by ANF. A 2-fold stimulation of the enzyme was obtained with 100 nM ANF and a half-maximal stimulation, with a 5 nM dose. The stimulation by ANF persisted for at least 30 min. Various detergents, such as Triton X-100, Lubrol PX, cholate, CHAPS, digitonin and zwittergent, stimulated several-fold the activity of particulate guanylate cyclase. However, only Triton X-100 dispersed particulate guanylate cyclase without affecting its response to ANF. The dose-response curve of ANF stimulation of the particulate and the Triton X-100 dispersed enzyme was similar. The dispersion of a fully responsive guanylate cyclase to ANF will help us to uncover the type of interactions between guanylate cyclase and ANF. It will also be used as a first step for the purification of an ANF-sensitive particulate guanylate cyclase.     

Intracellular trafficking and metabolic turnover ofligand-bound guanylyl cyclase/atrial natriuretic peptide receptor-A into subcellular compartments

Atrial natriuretic peptide (ANP) is the first described member of the natriuretic peptide hormone family. ANP elicits natriuretic, diuretic, vasorelaxant and antiproliferative effects, important factors in the control of blood pressure homeostasis. One of the principal loci involved in the regulatory action of ANP is the guanylyl cyclase-linked ANP-receptor which has been designated as NPRA, also referred to as GC-A, whose ANP-binding efficiency and guanylyl cyclase activity vary remarkably in different target tissues. However, the cellular and molecular basis of these activities and the functional expression and regulation of NPRA are not well understood. The mature form of receptor resides in the plasma membrane and consists of an extracellular ligand-binding domain, a single transmembrane-spanning region, and intracellular protein kinase-like homology and guanylyl cyclase catalytic domains. In this review, emphasis has been placed on the interaction of ANP with NPRA, the ligand-mediated endocytosis, trafficking, and subcellular distribution of ligand-receptor complexes from cell surface to the intracellular compartments. Furthermore, it is implicated that after internalization, the ANP/NPRA complexes dissociate into the subcellular compartments and a population of receptor recycles back to the plasma membrane. This is an interesting area of research in the natriuretic peptide receptor field because there is currently debate over whether ANP/NPRA complexes internalize at all or whether cell utilizes some other mechanisms to release ANP from the bound receptor molecules. Indeed, controversy exist since it has been previously reported by default that among the three natriuretic peptide receptors only NPRC internalizes with bound ligand. Hence, from a thematic standpoint it is clearly evident that there is a current need to review this subject and provide a consensus forum that establishes the cellular trafficking, sequestration and processing of ANP/NPRA complexes in intact cells. Towards this aim the cellular life-cycle of NPRA will be described in the context of ANP-binding, internalization, metabolic processing, and/or inactivation, down-regulation, and degradation of ligand-receptor complexes in model cell systems.     

Effects of phospholipase A and triton x-100 on guanylate cyclase activity in mammary gland homogenates from mice.

The effects of a variety of agents on guanylate cyclase activity were tested in broken cell preparations of mammary glands from midpregnant mice. Of the agents tested, only phospholipase A, triton X-100, and an impure egg lysolecithin preparation enhanced the activity of guanylate cyclase in mammary gland homogenates; other agents, including sodium azide and phospholipase C, and purified egg lysolecithin had no effect. Phospholipase A increased the activity of guanylate cyclase in the 150,000 g pellet fractions of mammary gland homogenates, bud did not consistently enhance guanylate cyclase in the 150,000 g supernatant fractions. Phospholipase A did not appear to enhance guanylate cyclase activity by solublizing the enzyme from the 150,000 g pellet. Triton X-100, in contrast, appeared to act by solubilizing guanylate cyclase from the material present in the 150,000 g pellet. Triton X-100 increased by several fold guanylate cyclase activity in the tissue homogenates and the 150,000 g pellets, but did not consistently enhance enzyme activity in the 150,000 g supernatant. Triton X-100 had no effect on the apparent Km of guanylate cyclase.     

Characterization and regulation by protein kinase C of renal glomerular atrial natriuretic peptide receptor-coupled guanylate cyclase

The nature and regulation of atrial natriuretic peptide (ANP)-sensitive guanylate cyclase in rat renal glomerular membranes was examined. By affinity crosslinking techniques, three bands with apparent molecular masses of 180, 130 and 64 kDa were specifically labeled with [125I]ANP. A specific antibody to the 180 kDa membrane guanylate cyclase of rat adrenocortical carcinoma recognized a 180 kDa band on Western blot analysis of solubilized, GTP-affinity purified glomerular membrane proteins. The same antibody completely inhibited ANP-stimulated guanylate cyclase activity in glomerular membrane fractions. Partially purified protein kinase C inhibited ANP-stimulated guanylate cyclase activity in glomerular membrane fractions. It is concluded that a 180 kDa ANP-sensitive guanylate cyclase is present in glomerular membranes, and that this enzyme is inhibited directly by protein kinase C.     

Bifunctional atrial natriuretic peptide receptor (type A) exists as a disulfide-linked tetramer in plasma membranes of bovine adrenal cortex.

Type A atrial natriuretic peptide (ANP) receptor was demonstrated to be present as a tetramer in the bovine adrenal cortex. Type A ANP receptor is composed of two functional domains, namely extracellular ANP-binding and cytoplasmic guanylate cyclase domains, and generally considered to be present as a single polypeptide chain of about 140 kDa based on its primary structure deduced from the cDNA sequence and its SDS/PAGE profile under reducing conditions. Characterization of the type A receptor or receptor/cyclase under non-reducing conditions led to the discovery stated in the title. The type A ANP receptor was partially purified from bovine adrenal cortex membranes by Blue-Sepharose and GTP-agarose chromatography. SDS-PAGE analysis of the receptor preparation revealed that although under reducing conditions it migrated as a 140-kDa band, the mobility of the receptor was greatly retarded in the absence of reducing agents, suggesting that the type A ANP receptor is present as a disulfide-linked oligomer in its native state. Further analysis using SDS-polyacrylamide-agarose gels suitable for determining the sizes of high-molecular-weight proteins revealed that the oligomer has an Mr of 500,000-550,000. This result clearly indicates that the native form of the type A receptor is a tetramer composed of four 140-kDa disulfide-linked receptor/cyclase molecules.     

Solubilization of the [8-lysine]vasopressin receptor and adenylate cyclase from pig kidney plasma membranes.

Adenylate cyclase and the [8-lysine]vasopressin receptor were solubilized from pig kidney medulla membranes using the nonionic detergent Triton X-100. Optimal conditions for solubilization were under continuous stirring in a medium containing 0.5% (/v) Triton X-100, 100 mM Tris-HCl, pH 8, and 10 mM MgCl2. Both adenylate cyclase activity and [3H][8-lysine]vasopressin binding activity were recovered in a -26,000 X g supernatant of detergent-treated membranes. The yield of solubilized adenylate cyclase was nearly 100%. The soluble enzyme was no longer sensitive to antidiuretic hormone but was slightly activated by sodium fluoride. The affinity of the soluble receptor for [8-lysine]vasopresin was les than that of the membrane-bound receptor (mean apparent Km values, respectively 10(-7) M and 2 X 10(-8) M), however binding cooperativity was preserved. Hill coefficients were 1.42 for the soluble receptor and 1.50 for the membrane receptor. The soluble receptor discriminated as efficiently as did the membrane receptor between [8-lysine-a1vasopressin and oxytocin. The yield of spolubilized receptor was only 30% despite the fact that all binding activity had disappeared from the residual pellet of detergent-treated membranes. When the membranous receptors were occupied before solubilization and the latter was performed under conditions in which dissociation of the hormone-receptor comples is slow, i.e. at low temperature, 65% to 100% of the hormone-receptor complex was recovered in the soluble fraction. The soluble hormone-receptor complex partially dissociated on rewarming whereas the free hormone concentration was kept unchanged in the medium. The residual binding capacity, which was 30% of the initial value, was identical with that determined when the receptor was solubilized in free form before incubation with labeled hormone. It was concluded that (a) solubilization of the receptor molecules was complete, (b) during solubilization two forms of the receptor appear, of which only one is accessible to the hormone, (c) occupancy of the receptor by the hormone prevented the formation of the nonaccessible form, and (d) some component or components of the soluble fraction might be responsible for the loss in apparent affinity.     

Regulation of the atrial natriuretic peptide receptor by heat shock protein 90 complexes

Heat shock protein 90 (hsp90) is a chaperone required for the proper folding and trafficking of many proteins involved in signal transduction. We tested whether hsp90 plays a role as a chaperone for GC-A, the membrane guanylate cyclase that acts as a receptor for atrial natriuretic peptide (ANP). When cultured cells expressing recombinant GC-A were treated with geldanamycin, an inhibitor of hsp90 function, the ANP-stimulated production of cyclic GMP was inhibited. This suggested that hsp90 was required for GC-A processing and/or stability. A physical association between hsp90 and GC-A was demonstrated in coimmunoprecipitation experiments. Treatment with geldanamycin disrupted this association and led to the accumulation of complexes containing GC-A and heat shock protein 70 (hsp70). Protein folding pathways involving hsp70 and hsp90 include several pathway-specific co-chaperones. Complexes between GC-A and hsp90 contained the co-chaperone p50(cdc37), typically found associated with protein kinase.hsp90 heterocomplexes. GC-A immunoprecipitates did not contain detectable amounts of Hop, FKBP51, FKBP52, PP5, or p23, all co-chaperones found in hsp90 complexes with other signaling proteins. The association of hsp90 and p50(cdc37) with GC-A was dependent on the kinase homology domain of this receptor but not on its ANP-binding, transmembrane, or guanylate cyclase domains. The data suggest that GC-A is regulated by hsp90 complexes similar to those involved in the maturation of protein kinases.     

Convulsant/barbiturate activity on the soluble gamma-aminobutyric acid-benzodiazepine receptor complex

Cage convulsant t-butyl bicyclophosphoro[35S]thionate binding activity in rat brain membrane homogenates was solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]propane sulfonate (Chaps) and shown to co-purify with the benzodiazepine--gamma-aminobutyric acid (GABA) receptor complex on gel filtration and affinity chromatography. Whereas convulsant binding activity, but not GABA and benzodiazepine receptor binding, was eliminated by solubilization in other detergents like sodium deoxycholate or Triton X-100, or by addition of Triton X-100 to the extracts solubilized in the zwitterionic detergent, convulsant activity was not irreversibly lost or selectively unstable, but could be restored by exchanging the protein back into the detergent Chaps. The GABA-benzodiazepine receptor activity solubilized in Chaps alone, containing convulsant activity, and a sample in Chaps supplemented with Triton X-100 and lacking convulsant activity, did not differ in size as measured by gel filtration column chromatography or by radiation inactivation target size analysis. This suggests that convulsant binding activity does not require any additional protein subunits or other macromolecules nor any unique aggregation state relative to GABA and benzodiazepine receptor binding, and that all three activities reside on the same protein complex. As in intact brain, the target size for convulsant binding activity was 3-5 times that of benzodiazepine binding activity, suggesting that an oligomeric protein structure of the receptor complex with intact strong subunit interactions present in the native membrane environment is needed for convulsant activity, and that this and other properties are more preserved in Chaps than in other detergents.