Evidence from the use of monoclonal antibody probes for structural heterogeneity of the growth hormone receptor.

We describe the use of four monoclonal antibodies (MAbs) to the rabbit liver growth hormone (GH) receptor and one raised against purified rat liver GH receptor to characterize liver receptor subtypes which differ in their hormone-binding regions. The anti-(rat liver GH receptor) MAb both inhibited and precipitated rat and rabbit GH receptors, but only one-half of 125I-oGH (ovine GH) binding to liver microsomes could be inhibited by excess antibody. Conversely, only one-half of 125I-anti-(rat GH receptor) MAb binding was inhibited by excess oGH and Scatchard plots for this MAb exhibited two components. Although only 50% of 125I-oGH binding to membranes was inhibited by this MAb, all solubilized receptor could be immunoprecipitated. We postulate two epitopes for the anti-(rat GH receptor) MAb, one located at the hormone-binding site (inhibitory site) and one elsewhere (immunoprecipitating site). A second, rabbit-specific antibody (MAb 7) inhibited 85% of hormone binding but only 30% of 125I-anti-(rat GH receptor) MAb binding to rabbit liver microsomes. A combination of this MAb with the anti-(rat GH receptor) MAb totally inhibited 125I-oGH binding. MAb 7 alone totally inhibited 125I-rat GH binding to rabbit liver microsomes, as it did with 125I-oGH binding to purified receptor. On the basis of these results and others we postulate three types of GH receptor in rabbit liver membranes and ascribe approximate extents of 125I-oGH binding to each. A cytosolic 'GH receptor' which is not poly(ethylene glycol)-precipitable is shown to share five epitopes with 'type 2' microsomal receptors. Purified plasma membrane and endoplasmic reticulum fractions derived from a rabbit liver microsomal preparation have identical antigenic characteristics with respect to the GH-binding region, indicating that the heterogeneity we describe is not related to receptor processing. Of the three types of GH receptor in the plasma membrane of the rabbit (and possibly rat) we postulate that one (type 1) corresponds to the GH receptor involved in stimulating growth and possesses all of the epitopes studied here. A second (type 2) appears to be identical with the cytosolic 'GH receptor' and lacks the epitope for the anti-(rat GH receptor) MAb in the hormone binding site region. A third (type 3) does not possess the epitope for the inhibitory anti-(rabbit GH receptor) MAb, appears not to bind rat GH and is lost during purification. The availability of type-specific MAbs will facilitate assignment of specific functions to liver receptor subtypes which mediate the multiple functions of GH.     

Growth hormone promoted tyrosyl phosphorylation of growth hormone receptors in murine 3T3-F442A fibroblasts and adipocytes

Because many growth factor receptors are ligand-activated tyrosine protein kinases, the possibility that growth hormone (GH), a hormone implicated in human growth, promotes tyrosyl phosphorylation of its receptor was investigated. 125I-Labeled human GH was covalently cross-linked to receptors in intact 3T3-F442A fibroblasts, a cell line which differentiates into adipocytes in response to GH. The cross-linked cells were solubilized and passed over a column of phosphotyrosyl binding antibody immobilized on protein A-Sepharose. Immunoadsorbed proteins were eluted with a hapten (p-nitrophenyl phosphate) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The eluate from the antibody column contained an Mr 134,000 125I-GH-receptor complex. A similar result was obtained when the adipocyte form of 3T3-F442A cells was used in place of the fibroblast form. O-Phosphotyrosine prevented 125I-GH-receptor complexes from binding to the antibody column, whereas O-phosphoserine and O-phosphothreonine did not. In studies of GH-promoted phosphorylation in 3T3-F442A fibroblasts labeled metabolically with [32P]Pi, GH was shown to stimulate formation of a 32P-labeled protein which bound to immobilized phosphotyrosyl binding antibodies. The molecular weight of 114,000 obtained for this protein is similar to that expected for non-cross-linked GH receptor. The Mr 114,000 phosphorylated protein could be immunoprecipitated with anti-GH antibody, indicating that GH remained noncovalently bound to this protein during absorption to and elution from the immobilized phosphotyrosyl binding antibody. Phosphoamino acid analysis after both limited acid hydrolysis and extensive base hydrolysis of the Mr 114,000 phosphoprotein confirmed the presence of phosphotyrosyl residues.(ABSTRACT TRUNCATED AT 250 WORDS)     

The specificity of binding of growth hormone and prolactin to purified plasma membranes from pregnant-rabbit liver.

The binding of 125I-labelled human growth hormone (hGH) to a purified plasma membrane preparation from the liver of pregnant rabbit, and to receptors solubilized from this fraction with Triton X-100, was dependent on time, temperature, the cations used and the receptor concentration. Solubilization did not affect the binding properties of the receptors at low concentrations of Triton X-100. Some somatogenic hormones, such as bovine GH, and some lactogenic hormones, such as ovine prolactin, displaced 125I-labelled hGH from purified plasma membranes and solubilized receptor preparations, but GHs and prolactins from various other species were rather ineffective. The results indicate that although there are binding sites for hGH in these pregnant rabbit liver membranes, few of these are specifically somatogenic or lactogenic. The binding properties of the purified plasma membranes are similar to those of a microsomal preparation studied previously, suggesting that the complex nature of the binding of hGH is not due to the heterogeneity of cellular membranes used to study binding, but is a property of the receptors associated with plasma membranes.     

Solubilization and characterization of CCK receptors from mouse pancreas

To study the characteristics of the CCK receptor, plasma membranes were prepared from mouse pancreatic acini, and CCK receptors solubilized with 1% digitonin. To measure hormone binding, the solubilized receptors were incubated with 125I-CCK at 4 degrees C and the hormone-receptor complex was precipitated with 10% polyethylene glycol. Specific 125I-CCK binding by the solubilized CCK receptor was compared to that by the plasma membrane-bound CCK receptor. Both the solubilized and the membrane-bound receptor displayed optimal binding at an acidic pH (between 6.0 and 7.0) and showed a similar sensitivity to monovalent and divalent cations. The solubilized receptors preserved their relative specificity for CCK molecules: CCK-8 greater than CCK-33 greater than desulfated CCK-8 greater than CCK-4. However, the soluble CCK receptor had a lower binding affinity than plasma membrane-bound receptor. Solubilized receptors preserved their relative specificity for inhibitors of CCK binding and action: dibutyryl cyclic GMP greater than N-CBZ-tryptophan greater than proglumide. Solubilized receptors had affinities for these antagonists that were similar to receptors on intact plasma membranes. These data indicate, therefore, that the specific binding properties of the CCK receptor are inherent to the solubilized glycoprotein molecules.     

Binding properties of solubilized gonadotropin-releasing hormone receptor: role of carboxylic groups

The interaction of 125I-buserelin, a superactive agonist of gonadotropin-releasing hormone (GnRH), with solubilized GnRH receptor was studied. The highest specific binding of 125I-buserelin to solubilized GnRH receptor is evident at 4 degrees C, and equilibrium is reached after 2 h of incubation. The soluble receptor retained 100% of the original binding activity when kept at 4 or 22 degrees C for 60 min. Mono- and divalent cations inhibited, in a concentration-dependent manner, the binding of 125I-buserelin to solubilized GnRH receptor. Monovalent cations require higher concentrations than divalent cations to inhibit the binding. Since the order of potency within the divalent cations was identical with that of their association constants to dicarboxylic compounds, it is suggested that there are at least two carboxylic groups of the receptor that participate in the binding of the hormone. The carboxyl groups of sialic acid residues are not absolutely required for GnRH binding since the binding of 125I-buserelin to solubilized GnRH receptor was only slightly affected by pretreatment with neuraminidase and wheat germ agglutinin. The finding that polylysines stimulate luteinizing hormone (LH) release from pituitary cell cultures with the same efficacy as GnRH suggests that simple charge interactions can induce LH release. According to these results, we propose that the driving force for the formation of the hormone-receptor complex is an ionic interaction between the positively charged amino acid arginine in position 8 and the carboxyl groups in the binding site.     

alpha 2-Macroglobulin binding to cultured fibroblasts. Solubilization and partial purification of binding sites

Binding sites having the characteristics of receptors for "activated" alpha 2-macroglobulin (alpha 2M) have been solubilized with octyl-beta-D-glucoside from fibroblast membranes. When the detergent was removed by dialysis, the resulting insoluble extract was shown to bind 125I-alpha 2M specifically. Analysis of the binding data using a nonlinear curve-fitting program suggests that the solubilized preparation contains two classes of binding sites (KD = 0.34 nM and KD = 104 nM). Membranes or solubilized extracts from KB cells which lack alpha 2M binding sites did not specifically bind 125I-alpha 2M. The solubilized binding sites from fibroblasts were inactivated by boiling and trypsin treatment, and required Ca+2 for maximal binding. In addition, the high affinity binding of 125I-alpha 2M to the solubilized receptor was inhibited by bacitracin and by alpha-bromo-5-iodo-4-hydroxy-3-nitroacetophenone, two agents which interfere with the uptake of alpha 2M in cultured fibroblasts. Using a combination of ion exchange and gel permeation chromatography, we have purified the high affinity alpha 2M binding site approximately 100-fold from membrane derived from NIH-3T3 (spontaneously transformed) fibroblasts grown as tumors in mice. The receptor is apparently an acidic protein and the receptor octyl-beta-D-glucoside complex has a Stokes radius of 45-50 A as measured by gel filtration.     

Cholecystokinin induces the interaction of its receptor with a guanine nucleotide binding protein

To evaluate the relation between the pancreatic cholecystokinin (CCK) receptor and guanine nucleotide-binding protein(s) we studied the effects of nucleotides on 125I-CCK binding to pancreatic acinar plasma membranes, 125I-CCK binding to solubilized 125I-CCK receptors, and the stability of the solubilized 125I-CCK-receptor complex. In plasma membranes, guanine nucleotides both inhibited CCK binding and increased the dissociation of CCK from its receptor. The potency of the nucleotides studied was GTP gamma S = GMP-PNP greater than GTP much greater than ATP. When membranes were solubilized with digitonin, subsequent binding of CCK was insensitive to guanine nucleotides including GTP, GMP-PNP and GTP gamma S. However, if CCK binding occurred before solubilization of the membranes, guanine nucleotides increased dissociation at concentrations and with a specificity similar to that observed for effects on intact pancreatic membranes. It is concluded that guanine nucleotides act via a protein which is separable from the receptor to induce dissociation of bound CCK. Moreover, CCK binding induces an association in the plasma membrane of the CCK receptor with this guanine nucleotide binding protein.     

A ligand-receptor binding assay by receptor immobilization

Using transferrin-transferrin receptor binding as a model of ligand-receptor binding, we have developed a new and simple binding assay for the solubilized receptor. Solubilized membrane proteins containing transferrin receptor were immobilized by covalent binding to beads having chemical reactive epoxide groups, and then 125I-labeled transferrin was added to the beads. Dose-dependent, ligand-specific, and saturable binding of 125I-labeled transferrin to the immobilized membrane proteins were demonstrated and a Scatchard analysis derived affinity of Kd = 1.8 X 10(-9) M was obtained. These results indicate that the immobilization of receptors onto beads may be useful in a simple binding assay of the solubilized receptor.     

Solubilization of membrane receptor for epidermal growth factor.

The membrane receptor for epidermal growth factor (EGF) has been solubilized from A-431 tumor cells using Triton X-100. Operational criteria used to define solubilization include failure of the binding activity to be pelleted after centrifugation at 90,000 x g for 1.5 hrs and the requirement for polyethylene glycol precipitation to detect ¹²⁵I-EGF: receptor complexes on membrane filters. Properties of the solubilized EGF are characterized and compared to the properties of the particulate receptor. The specific binding capacity of the solubilized EGF receptor was 8.0 picomoles ¹²⁵I-EGF bound per mg protein--approximately 60% of the binding capacity of particulate receptor preparations. Also, solubilization of the EGF receptor resulted in a 10-fold decrease in the affinity of the receptor for ¹²⁵I-EGF.     

Solubilization of the active vasoactive intestinal peptide receptor from human colonic adenocarcinoma cells

The non-ionic detergent n-octyl-beta-D-glucopyranoside was used to solubilize the VIP (vasoactive intestinal peptide) receptor from human colonic adenocarcinoma cell line HT29-D4. The binding of monoiodinated 125I-VIP to the solubilized receptor was specific, time-dependent, and reversible. Scatchard analysis of data obtained from competitive displacement of monoiodinated 125I-VIP by native VIP suggested the presence of two classes of VIP binding sites with Kd values of 0.32 and 46.7 nM. The binding capacities of these two classes were 1.7 x 10(10) and 30.2 x 10(10) sites/mg of proteins, respectively. The solubilized receptor retained the specificity of the human VIP receptor towards the peptides of the VIP/secretin/glucagon family. The order of potency in inhibiting monoiodinated 125I-VIP binding was VIP (IC50 = 1.0 x 10(-9) M) much greater than peptide histidine methionine amide (IC50 = 10(-7) M) greater than growth hormone-releasing factor (IC50 = 3 x 10(-7) M) greater than secretin (IC50 greater than 10(-6) M); glucagon had no effect on VIP binding. The reducing agent dithiothreitol inhibited in a dose-dependent manner the binding of 125I-VIP. Covalent cross-linking experiments between the solubilized receptor and 125I-VIP showed that after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography two major and one minor polypeptides of Mr 67,000, 72,000, and 83,000 were specifically labeled. When analyzed by gel filtration, the n-octyl-beta-D-glucopyranoside-solubilized 125I-VIP-receptor complex was resolved into two major peaks with molecular mass in the range of 60-70 and 270-300 kDa. Thus, the soluble form of the VIP receptor was probably a multimeric complex in which disulfide bonds may play an important role to hold the receptor in an active configuration.     

Isolation of a gastrin releasing peptide receptor from normal rat pancreas.

The presence of a putative GRP receptor on rat pancreatic particulate membranes was demonstrated by covalent cross-linking to 125I-gastrin releasing peptide (GRP), which revealed a radioactive band with Mr = 80-90 kDa on reduced SDS-PAGE. Fresh rat pancreatic membranes contained a GRP receptor which was solubilized with Triton X-100 as assessed by its failure to sediment at 100,000 x g for one hour and its ability to pass through a 0.22 mu filter. When 125I-GRP binding was studied using Sephadex G50 gel filtration chromatography to separate bound from unbound ligand, substantial amounts of 125I-GRP binding were observed in rat crude solubilized pancreatic membranes, but essentially no specific binding was observed until the crude solubilized membranes were fractionated by ammonium sulfate precipitation. Specific 125I-GRP binding was 500, 700 and 1400 fmol/mg protein, respectively, in the 0-25%, 25-50% and 50-80% saturated ammonium sulfate fractions (125I-GRP concentration = 1 nM). Specific binding was temperature dependent, saturable and of high affinity, (KD = 2.3 nM). A unique 70 kDa band was visualized by silver staining of the SDS-PAGE of eluates of GRP(14-27) affinity gel compared with eluates of control affinity gels incubated with the 25-50% (NH4)2SO4 fraction. The lower Mr than that observed with covalent cross-linking may represent the binding subunit of a larger receptor protein. This ligand-affinity isolated protein is thus a good candidate for the GRP receptor, or the binding subunit of it, from normal rat pancreas.     

Solubilization of the 17 alpha-ethinyl estradiol-stimulated low density lipoprotein receptor of male rat liver

Pharmacological doses of 17 alpha-ethinyl estradiol induce a low density lipoprotein (LDL) receptor in the liver of male rats. Our aim was to solubilize this receptor. Isolated liver membranes (8,000-100,000 g fraction) from male rats treated with 17 alpha-ethinyl estradiol and from control rats were solubilized in 1% (w/v) Triton X-100. Using Amberlite XAD-2, more than 90% of the detergent was then removed. Liposomes were prepared by precipitating the solubilized proteins with acetone in the presence of phosphatidylcholine. The receptor activity of these liposomes was assayed using human 125I-labeled LDL. Filtration was used to separate bound from free 125I-labeled LDL. The assay was optimized; 0.25 mM CaCl2, 25 mM NaCl, pH 8.0, were chosen as the standard conditions. Binding of 125I-labeled LDL was dependent on Ca2+. Liposomes containing solubilized membrane proteins from treated rats displayed Ca2+-dependent binding which was 11 times higher than for control rats. The specific binding of 125I-labeled LDL was saturable with a Kd = 18 micrograms/ml. 125I-Labeled LDL was displaced by unlabeled lipoproteins containing apolipoproteins B and E and by dimyristoylphosphatidylcholine liposomes containing purified apolipoprotein E, but not by HDL3. The binding was abolished by pronase and was inhibited by suramin. Ligand blotting with 125I-labeled LDL revealed one band of protein with an apparent molecular weight of 133,000 daltons. These properties are characteristic of the low density lipoprotein receptor.     

Solubilization of receptors for thyrotropin-releasing hormone from GH4C1 rat pituitary cells: demonstration of guanyl nucleotide sensitivity

TRH receptors have been solubilized from GH4C1 cells using the plant glycoside digitonin. Solubilized receptors retain the principal binding characteristics exhibited by the TRH receptor in intact pituitary cells and their membranes. The binding of the methylhistidyl derivative of TRH [( 3H]MeTRH) attained equilibrium within 2-3 h at 4 C, and it was reversible, dissociating with a t1/2 of 7 h. Analysis of [3H]MeTRH binding to soluble receptors at 4 C yielded a dissociation constant (Kd) of 3.8 nM and a total binding capacity (Bmax) of 3.9 pmol/mg protein. Peptides known to interact with non-TRH receptors on GH cells failed to interfere with the binding of [3H]MeTRH, indicating that the TRH binding was specific. Chlordiazepoxide, a competitive antagonist for TRH action in GH cells, inhibited TRH binding to soluble receptors with an IC50 of 11 microM. When [3H]MeTRH was bound to membranes and the membrane proteins were then solubilized, we found enhanced dissociation of the prebound [3H]MeTRH from its solubilized receptor by guanyl nucleotides. Maximal enhancement of [3H]MeTRH dissociation by 10 microM GTP gamma S occurred within about 45 min at 22 C. GTP gamma S, GTP, GDP beta S, and GDP were all effectors of [3H]MeTRH dissociation, exhibiting EC50s in the range of 14-450 nM. The rank order of potency of the tested nucleotides was GTP gamma S greater than GTP congruent to GDP beta S greater than GDP much greater than ATP gamma S greater than GMP. We conclude that TRH receptors have been solubilized from GH cells with digitonin and retain the binding characteristics of TRH receptors in intact pituitary cells. Furthermore, prebinding [3H]MeTRH to GH4C1 cell membranes results in the solubilization of a complex in which the TRH receptor is linked functionally to a GTP binding protein.     

Sedimentation behavior of solubilized gonadotropin receptor from plasma membranes of bovine corpus luteum.

The gonadotropin receptors associated with plasma membrane fractions were solubilized by detergents, including Triton X-100, Lubrol WX, Lubrol PX and sodium deoxycholate before and after equilibration with 125I-labelled human chorionic gonadotropin. The binding activity remained in solution even after centrifugation at 300 000 X g for 3 h. The solubilized gonadotropin receptor or gonadotropin receptor complex was characterized by gel filtration and sucrose density gradient centrifugation. Sucrose density gradient centrifugation of solubilized gonadotropin-receptor complex in the presence of Triton X-100 had a sedimentation coefficient of 6.5 S whereas the solubilized uncomplexed receptor had a sedimentation coefficient of 5.1 S. In the absence of the detergent, solubilized hormone receptor complex from plasma membrane fractions I and II sedimented with an apparent sedimentation coefficient of 6.6 S and 7.4 S, respectively. Similarly, the free receptor also showed higher sedimentation profile with an apparent sedimentation coefficient of 6.7 S for fraction I and 7.2 S for fraction II. Treatment of plasma membranes with phospholipase A and C inhibited the binding of 125I-labelled human chorionic gonadotropin in a dose dependent manner, whereas phospholipase D was without any effect. Doses of 1.4 mI. U. of phospholipase A or 0.6 mI.U. of phospholipase C were required to produce 50% inhibition of the binding activity. These phospholipases had no effect on the preformed 125I-labelled human chorionic gonadotropin-receptor complex nor on the sedimentation profile of solubilized gonadotropin receptor complex.     

Solubilization, gel filtration and sedimentation behaviour of prolactin receptors from human ovarian tissue

Receptors for 125I-labelled human prolactin have been identified in the crude membrane fraction isolated from human ovarian tissue. The non-ionic detergent Triton X-100, has been used to solubilize the membrane fraction. The presence of the receptor in the detergent extract was demonstrated by gel filtration and sucrose density gradient centrifugation. The binding was time-temperature dependent, being maximal at 23 degrees C after 15 h of incubation. Large amounts of other peptide hormones did not inhibit the binding of 125I-labelled human prolactin. The binding Scatchard analysis demonstrated that the affinity of the soluble receptor (Ka 1.13 +/- 0.15 X 10(10) M-1) for the labelled hormone was slightly greater than that of the crude membrane fraction (Ka 0.91 +/- 0.12 X 10(10) M-1). The binding capacity of the solubilized receptor was also significantly greater than that seen in the particulate before solubilization. The apparent Stokes radius of the solubilized receptor was estimated to be 57 A and that the hormone-receptor complex 60 A. The sedimentation coefficient of the solubilized receptor was 7.0 +/- 0.1 s, whereas that of the hormone-receptor complex was 7.5 +/- 0.2 s.     

VIP receptors from porcine liver: high yield solubilization in a GTP-insensitive form.

Vasoactive intestinal peptide (VIP) receptors were solubilized from porcine liver membranes using CHAPS. The binding of 125I-VIP to solubilized receptors was reversible, saturable and specific. Scatchard analysis indicated the presence of one binding site with a Kd of 6.5 +/- 0.3 nM and a Bmax of 1.20 +/- 0.15 pmol/mg protein. Solubilized and membrane-bound receptors displayed the same pharmacological profile since VIP and VIP-related peptides inhibited 125I-VIP binding to both receptor preparations with the same rank order of potency e.g. VIP greater than helodermin greater than rat GRF greater than rat PHI greater than secretin greater than human GRF. GTP inhibited 125I-VIP binding to membrane-bound receptors but not to solubilized receptors supporting functional uncoupling of VIP receptor and G protein during solubilization. Affinity labeling of solubilized and membrane-bound VIP receptors with 125I-VIP revealed the presence of a single molecular component with Mr 55,000 in both cases. It is concluded that VIP receptors from porcine liver can be solubilized with a good yield, in a GTP-insentive, G protein-free form. This represents a major advance towards the purification of VIP receptors.     

Identification of gonadotropin inducible, high density lipoprotein receptors in the solubilized membranes from rat ovary

Specific receptors for high density lipoproteins (HDL3) were solubilized from membranes of rat corpus luteum using different detergents. Among the detergents tested, octyl-beta-D-glucoside (40 mM) was most effective with respect to recovery of binding activity. The receptor activity released into 105,000 X g supernatant, can be assayed directly or with the precipitate obtained after dilution of the soluble supernatant. The 125I-HDL3 binding activity in the precipitated extract was linear with time, proportional to the amount of protein in the incubation mixture and saturable with increasing concentrations of 125I-HDL3. The solubilized receptor has an equilibrium dissociation constant (Kd) of 21.2 micrograms/ml and the binding activity was insensitive to Ca+2, EDTA and NaCl. These properties are similar to the membrane associated receptor. Administration of gonadotropin induced the HDL3 receptor in the solubilized membranes, suggesting that this receptor represents the physiologic receptor in the ovary.     

Solubilization and Partial Characterization of Angiotensin II Receptors from Rat Brain

Rat brain angiotensin II (Ang II) receptors were solubilized with a yield of 30-40% using the synthetic detergent 3[(3-cholamidopropyl)dimethylammonio)]-1-propanesulfonate. Kinetic analysis employing the high-affinity antagonist 125I-Sar1,Ile8-Ang II indicated that the solubilized receptors exhibited the same properties as receptors present within intact brain membranes. Furthermore, there was a positive correlation (r = 0.99) between the respective pIC50 values of a series of agonist and antagonists competing for 125I-Sar1,Ile8-Ang II labeled binding sites in either solubilized or intact membranes. Moreover, covalent labeling of 125I-Ang II to solubilized receptors with the homo-bifunctional cross-linker disuccinimidyl suberate, followed by gel filtration, revealed one major and one minor binding peak with apparent molecular weights of 64,000 and 115,000, respectively. Two binding proteins of comparable molecular weights (i.e., 112,000 and 60,000) were also identified by covalent cross-linking of 125I-Ang II to solubilized brain membranes followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. In contrast, only the smaller molecular mass binding protein was observed when solubilized membranes were labeled with the antagonist 125I-Sar1,Ile8-Ang II prior to gel filtration, and chromatofocusing of antagonist labeled sites revealed only one peak with an isoelectric point of 6.2. The successful solubilization of these binding sites should facilitate continued investigation of Ang II receptors in the brain.     

Detergent solubilization of the interleukin 1 receptor

Interleukin 1 (IL 1) receptors were solubilized from membranes prepared from murine EL-4 thymoma cells with the zwitterionic detergent 3[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS). Binding of IL 1 to the solubilized receptor was detected by a polyethylene glycol (PEG) precipitation procedure. Concentrations of CHAPS from 4 to 8 mM were effective in solubilizing the IL 1 receptor. At 10 mM CHAPS, there was some loss in binding activity, whereas 2 mM CHAPS was completely ineffective in solubilizing the receptor. Detergent concentrations of 4 mM were routinely used. The solubilized receptor retains the ability to bind 125I-IL 1 in a specific and saturable manner. Scatchard analysis reveals a single type of high affinity binding site having an apparent dissociation constant (KD) of approximately 1.2 X 10(-10) M. Nearly identical KD values are observed for membrane fractions. There are approximately 400 to 500 fmol receptor/mg protein in the detergent extract, corresponding to a two- to threefold enrichment in the Bmax observed for membranes. There is no loss in receptor activity as determined by complete recovery of the total number of binding sites from membranes after solubilization. Binding kinetics show that apparent steady state for the solubilized receptor is reached after 60 min at 37 degrees C. The binding of 125I-IL 1 is essentially irreversible because relatively little bound ligand can be dissociated from the receptor on the addition of excess unlabeled IL 1 at 37 degrees C. Both human IL 1 alpha and IL 1 beta compete for binding of 125I-IL 1 to the soluble receptor, confirming that IL 1 alpha and IL 1 beta bind to the same receptor. Other recombinant proteins, including interferon-alpha A, interferon-gamma, and interleukin 2 have no inhibitory effect.     

The receptor site for the bee venom mast cell degranulating peptide. Affinity labeling and evidence for a common molecular target for mast cell degranulating peptide and dendrotoxin I, a snake toxin active on K+ channels

The mast cell degranulating peptide (MCD) and dendrotoxin I (DTXI) are two toxins, one extracted from bee venom, the other one from snake venom, that are thought to act on voltage-sensitive K+ channels. Binding sites for the two toxins have been solubilized. The solubilized sites were stable and retained their high affinity for 125I-DTXI and 125I-MCD (Kd approximately equal to 100 pM). Interactions were found between MCD and DTXI binding sites in the solubilized state, establishing that the two different toxins act on the same protein complex. This conclusion was strengthened by the observations (i) that conditions of solubilization that eliminated 125I-MCD binding activity also eliminated 125I-DTX binding activity while both types of activities were preserved in the presence of K+ or Rb+ and (ii) that binding components for the two types of toxins had similar sedimentation coefficients and copurified in partial purifications. A component of the receptor protein for 125I-MCD has been identified; it has a Mr of 77,000 +/- 2000. This polypeptide was similar to or identical in molecular weight with that which serves as a receptor for DTXI (Mr 76,000 +/- 2000).