Purification of the receptor for nerve growth factor from A875 melanoma cells by affinity chromatography

The receptor for nerve growth factor (NGF) has been purified to near homogeneity from octylglucoside extracts of A875 melanoma cell membranes by the use of repetitive affinity chromatography on NGF-Sepharose. Elution of purified receptor (NGF receptor) was accomplished with 0.15 M NaCl, pH 11.0, containing phosphatidylcholine and octylglucoside. Chromatography on two columns of NGF-Sepharose yielded a 1500-fold purification of the receptor, as assessed by 125I-NGF binding, and permitted recovery of 9% of the total binding activity in the soluble extract. Scatchard analysis of equilibrium binding of 125I-NGF provided similar Kd values for NGF receptors in soluble extracts of A875 membranes (2.2 nM) and with purified NGF receptor (3.1 nM). Examination of NGF receptor after electrophoresis on sodium dodecyl sulfate-polyacrylamide gels revealed the presence of two major peptides, of Mr = 85,000 and Mr = 200,000. Affinity labeling experiments, done with 125I-NGF and A875 cells, soluble extracts of A875 cell membranes, and purified receptor, show that both of these components of the NGF receptor can be specifically cross-linked to 125I-NGF.     

A comparison of binding properties and structure of NGF receptor on PC12 pheochromocytoma and A875 melanoma cells

Rat PC12 pheochromocytoma and human A875 melanoma cells express nerve growth factor (NGF) receptors on their surfaces. Covalent crosslinking of bound 125I-NGF to PC12 or A875 intact cells or plasma membrane-enriched fractions resulted in labelling of a peptide doublet at Mr = 110,000 and a single labelled peptide at Mr = 200,000 for each of the cell and membrane preparations. However, a difference between equilibrium binding properties of NGF-receptor on PC12 and A875 cells was observed. PC12 cells exhibited biphasic binding properties with two apparent binding sites: KD = 5.2 nM sites and KD = 0.3 nM sites. The high-affinity PC12 binding sites were trypsin resistant, and 125I-NGF dissociated slowly from them. A875 cells exhibited sites with homogeneous properties (KD = 1.0 nM), all binding sites were trypsin sensitive, and 125I-NGF dissociated rapidly in the presence of unlabelled NGF. Membrane-enriched fractions from either cell type contained binding sites with a uniform low affinity (KD = 3 nM) that were trypsin sensitive, and 125I-NGF rapidly dissociated from them. Sixty to 80 percent of binding sites in membranes could be converted to the high-affinity, trypsin-resistant state by addition of wheat germ agglutinin (WGA). The loss of high-affinity, trypsin-resistant sites from PC12 cells during preparation of plasma membrane fractions does not appear to be the result of selective isolation of low-affinity sites or proteolytic degradation since there is a loss of 125I-NGF binding immediately after cell lysis which is not blocked by protease inhibitors. Also, high-affinity, trypsin-resistant binding sites are not found associated with other cell fractions. The differences between receptor properties on PC12 cells and on A875 cells apparently are the result of differences in the respective intracellular environments. Thus, significant structural homology exists between receptors on A875 and PC12 cells. Cell components other than the binding unit of the NGF receptor may be responsible for the different properties of receptor.     

Purification of a 130-kDa T cell glycoprotein that binds human interleukin 4 with high affinity

The interleukin 4 (IL-4) receptor was purified from the gibbon T cell line MLA 144. These cells were found to express high numbers of human IL-4-binding proteins (5000-6000 sites/cell) with an affinity constant (Kd) similar to that measured in human cell lines (Kd = 40-70 pM). Affinity cross-linking of 125I-IL-4 to human cell lines and MLA 144 cells demonstrated the labeling of three proteins of approximately 130, 75, and 65 kDa. Human IL-4-binding sites were solubilized from MLA 144 cells using Triton X-100 and then purified by carboxymethyl chromatography, which removed 50% of the protein without loss of IL-4-binding activity. Then sequential affinity purification over wheat germ agglutinin and a single IL-4 Affi-Gel 10 column resulted in a final 8000-fold purification of the IL-4 receptor. When analyzed on a silver-stained sodium dodecyl sulfate-polyacrylamide gel, the purified receptor migrated as a single molecular species of 130 +/- 5 kDa. Identification of the 130-kDa protein as the IL-4 receptor was demonstrated by cross-linking experiments and specific binding of 125I-IL-4 to nitrocellulose membranes after electrophoretic transfer of the purified receptor on sodium dodecyl sulfate-polyacrylamide gel.     

Affinity chromatography of the bovine cerebral cortex A1 adenosine receptor

An approximate 140-fold purification of the A1 adenosine receptor of bovine cerebral cortex has been obtained via affinity chromatography. The affinity column consists of Affi-Gel 10 coupled through an amide linkage to XAC, a high-affinity A1 adenosine receptor antagonist. As assessed by [3H]XAC binding, bovine brain membranes solubilized with the detergent CHAPS had a specific binding activity of 1.1 pmol/mg protein. Interaction of solubilized A1 adenosine receptors with the XAC-Affi-Gel was biospecific and 30% of the receptor activity was bound by the gel. Demonstration of [3H]XAC binding in the material eluted from the column with R-PIA required insertion of receptor into phospholipid vesicles. The specific activity of the affinity column purified receptor was 146 +/- 22 pmol/mg protein with typically 5-15% of the bound receptor recovered. The purified receptor displayed high-affinity antagonist binding and bound agonists with the potency order expected of the bovine brain A1 adenosine receptor: R-PIA greater than S-PIA greater than NECA. In purified preparations, the photoaffinity probe [125I]PAPAXAC-SANPAH specifically labelled a protein of molecular mass 38,000 which has previously been shown to be the A1 adenosine receptor binding subunit.     

Characterization and regional distribution of calcitonin binding sites in the rat brain

Binding sites for calcitonin (CT), as assayed by the displacable binding of [125-I] iodo salmon CT ([125-I]sCT), were found on a membrane fraction prepared from rat brain. The half times of association varied between 23 and 7 min as a function of the temperatures used in the incubation medium, ranging from 6° to 37°C. Salmon CT in amounts as low as 10^?10 M inhibited the binding of [125-I]sCT to the membranes, whereas the virtually biologically inactive free acid of human CT and human CT sulfone did not affect the binding. The specific binding of [125-I]sCT to the membranes was directed to structural and/or conformational features in the COOH-terminal half of salmon CT. 133 to 8,900 times higher amounts of porcine CT and human CT and analogues thereof were required to achieve an inhibition of binding equal to that produced by salmon CT. Sixty-seven percent of specific binding of labeled hormone was not dissociable, even after 6 h of incubation with an excess of unlabeled hormone. [125-I]sCT extracted from the membranes was not degraded, as judged by gel permeation chromatography, and retained binding activity. Specific binding was highest in the hypothalamus, followed by the brainstem. It was intermediate in the midbrain-thalamus and the striatum, lower in the cortex and negligible in the hippocampus, and cerebellum and the spinal cord.     

Properties of receptors for epidermal growth factor in detergent solution: Evidence for heterogeneous aggregated states

Between 60% and 100% of epidermal growth factor (EGF) binding activity was recovered from membranes of the A431 human epidermoid carcinoma cell line treated with solutions containing the nonionic detergent Triton X-100. Approximately half of the recovered binding activity was sedimented at low centrifugal forece and hence was operationally insoluble in nonionic detergent solution. Receptors in both the detergent-soluble and -insoluble fractions displayed similar affinities for ¹²⁵I-EGF, and the values were in good agreement with those obtained for receptors in untreated membranes. The receptors in both fractions also formed identical direct linkage complexes with ¹²⁵I-EGF in similar yield, providing no evidence for partitioning of different molecular species of EGF receptors in the detergent-soluble and -insoluble fractions. Gel chromatography of the detergent-soluble membrane fraction on Sepharose 6-B revealed heterogeneity of ¹²⁵I-EGF binding activity; the smallest and most monodisperse peak of activity resolved by this technique was eluted at a Stokes radius of 95 Å. Operationally soluble ¹²⁵I-EGF binding activity also behaved heterogeneously during velocity sedimentation; more than half the activity sedimented more rapidly than the apparently monidisperse, 7S form. An average of less than half the nonionic detergent-solubilized activity recovered from 10 independent membrane preparations behaved as an apparently monodisperse entity. Since a maximum of 60% of ¹²⁵I-EGF binding activity was operationally soluble, less than 25% of the total EGF binding activity was recovered in an apparently monodisperse form. The remaining 75% of the EGF receptors displayed a marked tendency to exist as aggregates in nonionic detergent solutions.     

Solubilization and purification of a membrane-associated 3,3'',5-tri-iodo-L-thyronine-binding protein from rat erythrocytes.

3,3',5-Tri-iodo-L-thyronine (L-T3) binding sites from rat erythrocyte membranes were solubilized in an active form by using the zwitterionic detergent CHAPS or the anionic detergent lauroylsarcosine. The binding protein was successively purified by Sephadex G-200 and affinity chromatography. The purified material retained its binding activity and exhibited high affinity and specificity compared with those displayed in the original membrane. Yield was about 10% of the starting activity. The specific binding activity was enriched by approx. 100-fold, which represents a purity of only 0.1%. Analysis of the purified preparation on SDS/PAGE showed two major protein bands (Mr 64,000 and Mr 50,000), but these could not represent the binding protein since the purity obtained was low. However, affinity-labelling experiments with N-bromoacetyl-L-[125I]T3 in intact membranes showed that two proteins (also with Mr values of 64,000 and 50,000) bound the hormone specifically, suggesting a co-migration of hormone receptors and contaminants on gel electrophoresis.     

Purification and sequence determination of bovine atrial natriuretic factor

We report the purification and the sequence determination of bovine atrial natriuretic factor (ANF) in acid extracts of bovine atrial appendages. The monitoring of the activity along the purification steps was performed with a radio-receptor assay using bovine adrenal cortex membranes sites and 125I ANF. Bovine ANF was separated by carboxymethyl agarose gel chromatography from catecholamines and major protein contaminants. It behaved as a 3 K dalton peptide on Sephadex G-50. The active fractions were then subjected to high performance liquid chromatography (HPLC) using a sulfopropyl cation exchange column. Subsequent purification steps by reverse phase mode on Ultrapore RPSC, Vydac TP 218 and uBondapak using acetonitrile gradients led to the obtention of a pure fraction which amino acid sequence was identical to that for human ANF. This confirms the high degree of homology of ANF structure among mammalian species and advocates the use of the radio-receptor assay based on bovine adrenal receptor for measuring human ANF.     

Purification of basic fibroblast growth factor receptors from bovine brain

Fibroblast growth factors are proteins which play a major role, in vitro and in vivo, in the control of cellular growth and differentiation of a large number of cells. Biological activities of these factors are mediated by the interaction with specific membrane receptors. Previous studies indicated that the apparent molecular weight of a family of these receptors for the basic form of Fibroblast Growth Factor (bFGF), ranges from 125 to 165 kDa according to cell species and types. We have purified this family of receptors from bovine brain. We first set up a radioreceptor assay to detect receptors throughout the purification by measuring its ability to inhibit the fixation of radiolabeled bFGF to insolubilized membranes from bovine brain. The purification was also monitored by using cross-linking reagents in order to allow the visualization of radiolabeled bFGF bound to its receptor. The first purification steps involved 2 anion-exchange chromatographic steps, DEAE Trysacryl and FPLC Mono Q, and yielded an enrichment over 500 fold. Affinity chromatography with bFGF immobilized on Sepharose 4B was then performed. Covalent fixation of bFGF to the Sepharose matrix was carried out in presence of N-acetylated heparin in order to protect the recognition site for bFGF on its receptor. These 3 chromatographic steps yielded only 2 bands of apparent molecular weight of 100 kDa and 135 kDa as detected by electrophoresis. These 2 bands are also detected after chromatography on immobilized wheat germ agglutinin hence confirming the presence of carbohydrates on bFGF receptors.     

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.     

Purification and Characterization of B2 Bradykinin Receptor from Rat Uterus

Abstract: A B₂ bradykinin (BK) receptor was solubilised and partially purified from rat uterine membranes by a combination of ammonium sulphate precipitation, desalting on Sephadex G-50, and hydroxyapatite and wheat germ agglutinin affinity chromatography. The partially purified BK receptor, enriched 1,500-fold, was then cross-linked to ¹²⁵l-Tyr⁰-BK using disuccinimidyl suberate and purified to homogeneity as a single protein species on two-dimensional gel electrophoresis with a molecular mass of 81 kDa. This molecular size was in agreement with the value of 80–120 kDa estimated from Sephacryl 300 size exclusion column chromatography of the B₂ receptor. The partially purified and the crude solubilised B₂ BK receptor from rat uterus showed similar affinities for BK and the BK analogues iodo-Tyr⁰-BK, D-Phe⁷-BK, and des-Arg⁹-BK, indicating that the ligand binding specificity of the receptor had been retained during the purification procedures. The biochemical properties of the solubilised B₂ BK receptor correspond to those of a hydrophobic acidic glycoprotein (isoelectric focusing gave a value of 4.5–4.7) that binds specifically to wheat germ agglutinin but has no affinity for either concanavalin A or lentil lectin, suggesting the absence of terminal mannose or glucose residues.     

Two forms of the GABAA receptor distinguished by anion-exchange chromatography

The GABAA receptor complex was solubilized from rat brain membranes in Triton X-100, enriched by 1012-S affinity chromatography, and subjected to DEAE anion-exchange chromatography. Two forms were distinguished by their differential elution during this HPLC with a KCl gradient. They displayed similar [3H]muscimol- and [3H]flunitrazepam-binding characteristics, as well as [3H]flunitrazepam-binding inhibition by CL 218872. Rechromatography of these distinct ionic forms indicated that they were not in dynamic equilibrium during chromatography. Resolution of these two pharmacologically similar populations of GABAA receptor by anion-exchange HPLC suggests that they differ in charge densities, a condition which may reflect differing glycosylation or phosphorylation states of the complex.     

Contamination of an anion-exchange membrane by glutathione

Electrodialysis, which can separate electrolytes under mild conditions by using ion-exchange membranes, is a strong candidate for separation of GSH from yeast extracts, because GSH is unstable and easily oxidized forming a disulfide bond especially under alkali conditions. In this paper, sorption behavior of GSH on an anion-exchange membrane, in the pH 3–6 region that is expected to be the most preferable for its electrodialytic separation, was examined. Sorption of GSH on a Selemion-AMV anion-exchange membrane was accelerated as the pH of the membrane-contact solution increased, and there was a good correlation between the sorbed amounts and the molar fraction of monovalent anionic species of GSH. However, the amounts of GSH desorbed from the membrane by a NaCl desorbing solution were much lower than the initial sorbed amounts, and the difference between them was enlarged with increasing pH. The GSH which was lost could be recovered by the addition of DTT in the membrane-contact and desorbing solutions. Similar results were also obtained with Cys. We thus concluded that an anion-exchange membrane would be contaminated by thiol compounds, such as GSH and Cys, through oxidative binding of the thiol group with the membrane, the local OH^- concentration in which was enhanced due to attraction by the positively charged anion-exchange membrane.     

Binding of monoiodinated neuropeptide Y to hippocampal membranes and human neuroblastoma cell lines

Monoiodinated radioligands of the homologous 36-amino acid peptides, neuropeptide Y (NPY) and peptide YY, were prepared by reverse phase high performance liquid chromatography with isocratic elution. [125I-Tyr1]- and [125I-Tyr36]monoiodoNPY bound equally well to a single class of high affinity binding sites on synaptosomal membranes prepared from porcine hippocampus (Kd = 1.0 X 10(-10) M) whereas iodine substitution in Tyr27, for example, partly interfered with the receptor binding. The receptors on the hippocampal membranes did not distinguish between neuropeptide Y and peptide YY either in their monoiodinated or in their unlabeled forms. Six out of twelve human neuroblastoma cell lines had high affinity binding sites for monoiodinated NPY ranging from 2 to 145 X 10(3) sites per cell. The NPY binding to three of the cell lines, SMS-MSN, SMS-KAN, and CHP-234 was of relatively high affinity (Kd = 1.3 to 6.1 X 10(-10) M), and, as in the hippocampal membranes, the long C-terminal fragment, NPY(13-36)peptide was also a relatively potent ligand for these receptors. Two other neuroblastoma cell lines, MC-IXC and CHP-212, expressed NPY receptors characterized by a lower affinity (Kd = 4.8 and 24.6 X 10(-9) M) and negligible cross-reactivity with the C-terminal fragment. It is concluded that monoiodinated radioligands of the tyrosine-rich neuropeptide Y can be prepared and that receptors for these ligands in two apparently different subtypes are found on a series of human neuroblastoma cell lines.     

Solubilization of the Neuropeptide Y Receptor from Rat Brain Membranes

The neuropeptide Y (NPY) receptor was solubilized from rat brain membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS). The binding of 125I-NPY to CHAPS extracts was protein, time, and temperature dependent. Unlabeled NPY and the related peptides peptide YY (PYY) and pancreatic polypeptide inhibited 125I-NPY binding to solubilized receptors with relative potencies similar to those seen with membrane-bound receptors: NPY greater than PYY much greater than pancreatic polypeptide. Scatchard analysis of equilibrium binding data showed the CHAPS extracts to contain a single population of binding sites with a KD of 3.6 +/- 0.4 nM (mean +/- SEM) and a Bmax of 5.0 +/- 0.2 pmol/mg of protein. In addition the 125I-NPY binding to the soluble receptor was not inhibited by guanosine-5'-O-(3-thiotriphosphate), in contrast to the GTP sensitivity displayed by the membrane-bound receptor. Gel filtration chromatography using Sepharose 6B revealed a single peak of binding activity corresponding to a Mr of approximately 67,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis after chemical cross-linking revealed a single band at Mr 62,000. After solubilization and gel chromatography a 50- to 100-fold purification of the NPY receptor was obtained.     

Characterization of the binding properties and retrograde axonal transport of a monoclonal antibody directed against the rat nerve growth factor receptor

We have demonstrated in vitro and in vivo the specific binding of a monoclonal antibody to the rat nerve growth factor (NGF) receptor. Previous work had shown that this antibody, designated 192-IgG, does not compete with NGF for binding to the NGF receptor of PC12 cells, but instead interacts with the receptor to increase NGF binding to PC12 cells (Chandler, C. E., L. M. Parsons, M. Hosang, and E. M. Shooter, 1984, J. Biol. Chem., 259:6882-6889). In the present study, a solid-phase separation assay verified the specific formation of a ternary complex of 192-IgG, the NGF receptor, and NGF: 125I-labeled 192-IgG precipitated from solution only when incubated with both solubilized NGF receptor and NGF covalently linked to a solid phase (Sepharose 4B). Filtration assays using plasma membrane preparations of various tissues showed strict correlation of 125I-192-IgG and 125I-labeled NGF binding; only membranes obtained from superior cervical ganglion bound significant amounts of the monoclonal antibody and NGF. Injection of 125I-192-IgG into the rat anterior eye chamber led to accumulation of intact antibody molecules in the ipsilateral superior cervical ganglion, indicating retrograde axonal transport of 125I-192-IgG from the neuronal termini, located at the iris, to the cell bodies situated in the ganglion. The time course and saturation characteristics of 125I-192-IgG retrograde transport were very similar to those previously reported for 125I-NGF transport, indicating that 192-IgG can be internalized and transported by the same mechanisms as is NGF. Consistent with results of the in vitro binding assays, 192-IgG and NGF failed to compete for retrograde transport and were actually co-transported. Retrograde axonal transport of 192-IgG appears to be species specific, since 125I-192-IgG was transported in the rat, but not in mice, gerbils, hamsters, or guinea pigs. These results establish monoclonal antibody 192-IgG as a specific probe for the rat NGF receptor in vitro and in vivo.     

Biophysical characterization of the purified alpha 1-adrenergic receptor and identification of the hormone binding subunit

The alpha 1-adrenergic receptor has been solubilized in active form from rat hepatic membranes with the nonionic detergent, digitonin, and purified by affinity and gel filtration chromatography to homogeneity with a specific activity of 14,400 pmol/mg of protein. The affinity chromatographic steps of the purification procedure were achieved by the use of a newly synthesized analog (2-[4(2-succinoyl)piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline, CP-57,609) of the highly selective alpha 1-adrenergic antagonist, prazosin, immobilized via an amide linkage to agarose. The resulting purified receptor bound [3H]prazosin and a variety of adrenergic agents with the specificity, stereoselectivity, and affinities equivalent to those observed with membrane-bound and solubilized receptor preparations. The purified receptor.digitonin complex had a Stokes radius of 49 A and a sedimentation coefficient (s20w) of 7.1, as determined by AcA-34 gel filtration chromatography and sucrose gradient density centrifugation, respectively. Based on these hydrodynamic parameters, the calculated molecular weight of the receptor.digitonin complex was estimated at 147,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis following the final purification step revealed a single band of protein at 59,000 daltons from which [3H]prazosin binding activity could be recovered after renaturation of the receptor protein. These findings indicate that the protein purified from rat hepatic membranes is the hormone binding component of the alpha 1-adrenergic receptor and that the receptor molecule most likely contains more than one Mr = 59,000 subunit.     

Purification and chemical composition of a low molecular weight follicle-stimulating hormone binding inhibitor from porcine follicular fluid

Porcine follicular fluid contains several factors capable of inhibiting the binding, in vitro, of follicle-stimulating hormone (FSH) to receptor, including an agonist and an antagonist of FSH biological activity in vitro. FSH receptor-binding inhibitory activity (FSH-BI) was determined with assays using radioligand (125iodide-human FSH) receptor (calf-testes membrane); in vitro biological assays (cultured immature rat Sertoli cells) were used to determine antagonist/agonist activity. FSH antagonist activity is due to a low (less than 5000) molecular weight FSH-BI that is soluble in acidic acetone and insoluble in diethyl ether allowing preparative scale isolation. Additional purification was achieved by anion-exchange and reverse-phase high-performance liquid chromatography. Highly purified, biologically active FSH-BI contained the amino acids Ser, Gly, Arg, Thr, Ala, Pro, Val, and Lys; hexoses (phenol-sulfuric acid-positive reaction); and ethanolamine. Thus, this FSH antagonist appears to be a complex glycopeptide--possibly derived from membrane components, as suggested by the presence of ethanolamine and carbohydrate residues. Porcine follicular fluid, therefore, contains a low molecular weight FSH antagonist that, along with the high molecular weight FSH agonist previously identified, may regulate gonadal responsiveness to FSH through interactions with the FSH receptor.     

Purification and characterization of Leydig cell luteinizing hormone receptor

We have purified the testicular luteinizing hormone (LH/human choriogonadotropin (hCG)) receptor by sequential affinity chromatography on hCG-Sepharose. The purified LH/hCG receptor was identified as a single protein of Mr = 90,000 +/- 2,000 on sodium dodecyl sulfate-gel electrophoresis (SDS-PAGE), showed high affinity binding for hCG, and a binding capacity of 3.8 nmol/mg of protein. Electrophoretically blotted receptor retained the ability to bind 125I-hCG on nitrocellulose membrane, and the Mr of radioactive band was consistent with that revealed by silver staining. Autoradiography after SDS-PAGE analysis of cross-linked purified receptor-hCG complex showed Mr = 145,000 and Mr = 105,000 bands. These results are consistent with a Mr value for the receptor of 90,000 after accounting for contribution by the intact hormone or its alpha-subunit. Analysis of the free receptor by fast protein liquid chromatography on Superose 12 revealed a single peak of binding activity for 125I-hCG which eluted in the position of Mr = 200,000-240,000 in the presence of Triton X-100. Since a single protein species is observed under reducing or nonreducing conditions in SDS-PAGE, the receptor could exist in the membrane as a dimeric form composed of subunits Mr = 90,000 associated through noncovalent interactions. The pure receptor can be phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase (approximately 0.3 mol of phosphate/mol of receptor). This phosphorylation does not affect the binding characteristics of the receptor. The method described is simple and allows rapid purification of microgram amounts of biological active Leydig cell LH/hCG receptor for structural, functional, and immunological studies.     

Large-scale purification of beta-adrenergic receptors from mammalian cells in culture

S49 Mouse lymphoma wild-type cells were grown in spinner cultures of 40 liters to a density of approximately 3 million cells/ml. Growth of cells to high density (2-3 million cells/ml) required that the cell suspensions be bubbled with oxygen. Cells from 40 liter cultures were collected by centrifugation and disrupted by nitrogen cavitation. Highly purified membranes (0.35 g membrane protein) that were rich in beta-adrenergic receptor (0.4-0.7 pmol receptor/mg membrane protein) were prepared by differential centrifugation and then solubilized with the plant glycoside, digitonin (1.5% digitonin at 3 mg of membrane protein/ml). Beta-adrenergic receptors were isolated and purified by sequential affinity chromatography, ion-exchange chromatography, and steric exclusion high-pressure liquid chromatography. The extract was subjected to affinity chromatography on a derivatized Sepharose-4B CL column to which the high-affinity, beta-adrenergic antagonist (-)alprenolol had been immobilized. Following extensive washing, the receptor bound to this matrix was eluted using a 0-100 micromolar linear gradient of (-)alprenolol. The receptor eluted as a sharp peak at 30 micromolar ligand and displayed a specific activity of 280 pmol receptor/mg of protein. Ion-exchange chromatography on DEAE-Sephacel increased the specific activity to 950 pmol/mg of protein. The final step in the purification, steric-exclusion high-pressure liquid chromatography on two TSK-3000 and one TSK-2000 columns, tandem linked, resulted in a beta-adrenergic receptor preparation with a specific activity of 6700 pmol/mg of protein (15,900-fold purification). Autoradiography of the radioiodinated pure receptor, the receptor photolabeled with [125I]iodoazidobenzylpindolol or silver-staining of chemical amounts of protein revealed that the Mr of the pure receptor is 66,000 upon polyacrylamide gel electrophoresis in sodium dodecyl sulfate under reducing conditions. The receptor is a beta2-subtype adrenergic receptor.