Solubilization and hydrodynamic characterization of the dihydropyridine receptor from rat ventricular muscle

The dihydropyridine receptor-calcium channel complex, prelabeled with (+)-[3H]PN200-110, was solubilized from rat heart membranes with a detergent mixture of digitonin and Triton X-100. The dissociation of (+)-[3H]PN200-110 was slow enough to permit the hydrodynamic characterization of the complex by means of sucrose gradient sedimentation and gel filtration. The hydrodynamic properties of the complex were determined in several detergents, including Tween 80, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), and digitonin. S20,w values of 12.5, 15.4, and 21.0 S were obtained in sucrose gradients prepared in Tween 80, CHAPS, and digitonin, respectively. A Stokes radius of 86-87 A was obtained in each of the three detergents. Determination of the partial specific volume of the protein-detergent complex in each case revealed that the differences in S20,w values could be explained by the differences in the properties of the bound detergent species. Partial specific volumes of 0.796, 0.730, and 0.730 ml/g, corresponding to molecular weights of 595,000, 540,000, and 740,000 were obtained for the complex in Tween 80, CHAPS, and digitonin, respectively. This indicated that Tween 80 readily exchanged for the solubilizing mixture of digitonin and Triton X-100, whereas CHAPS did not. Detergent exchange with Tween 80 made it possible to determine the fractional contribution of the receptor protein to the molecular weight of the protein-detergent complex. The molecular weight of the dihydropyridine receptor-calcium channel complex was estimated to be 370,000. The protein-detergent complex was found to have a frictional coefficient of 1.39, consistent with a large transmembrane protein.     

Solubilization and affinity purification of the Y2 receptor for neuropeptide Y and peptide YY from rabbit kidney.

Neuropeptide Y (NPY) is an important neuropeptide in both central and peripheral neurones whereas peptide YY (PYY) is a gut hormone present in endocrine cells in the lower bowel. Both peptides interact with multiple binding sites that have been further classified into Y1 and Y2 receptors. We have solubilized native Y2 receptors both from basolateral membranes of proximal convoluted tubules from rabbit kidney and from rat hippocampal membranes. Solubilization of functional Y2 receptors was obtained with both 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and digitonin and resulted in each case in a single class of high affinity binding sites. The soluble receptor retained the binding specificity for different peptides and long C-terminal fragments of NPY exhibited by membrane preparations. Gel filtration of solubilized receptors resulted in a single peak of specific PYY binding activity corresponding to Mr = 350,000 whereas affinity labeling revealed a major band of Mr = 60,000. Since this binding activity was inhibited by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) the Y2 receptor is probably solubilized as a receptor complex containing a G-protein along with the ligand binding protein. Y2 receptor binding sites from kidney tubular membranes were purified to homogeneity by a three-step procedure employing Mono S cation-exchange adsorption, affinity chromatography on wheat germ lectin-agarose beads, and affinity chromatography on NPY-Affi-Gel. Electrophoresis and silver staining of the final receptor preparation revealed a single protein with Mr = 60,000 whereas gel filtration showed a single peak at approximately Mr = 60,000. The purified protein can be affinity labeled with [125I-Tyr36]PYY, indicating that the Mr = 60,000 protein contains the ligand binding site of the Y2 receptor, and this binding is not affected by GTP gamma S. Scatchard transformation of binding data for the purified Y2 receptors was compatible with a single class of binding sites with Kd = 76 pM. The purified Y2 receptors retain their binding properties with regard to affinity and specificity for different members of the pancreatic polypeptide-fold peptide family. The specific activity of purified Y2 receptors was calculated to approximately 14.7 nmol of ligand binding/mg of receptor protein, which is consistent with the theoretical value (16.6 nmol/mg) for a pure Mr = 60,000 protein binding one PYY molecule. Purification to homogeneity thus reveals the Y2 receptor as an Mr = 60,000 glycoprotein.     

Y2 receptor proteins for peptide YY and neuropeptide Y. Characterization as N-linked complex glycoproteins.

Affinity labeling using [125I-Tyr36]PYY and homobifunctional affinity crosslinking reagents of the rabbit Y2 receptor for peptide YY(PYY) results in specifically labeled proteins of both M(r) = 50,000 to 60,000 and M(r) = 96,000 to 115,000 [1,2]. In this work the glycoprotein nature of affinity labeled Y2 receptor proteins were investigated by enzymatic deglycosylation using neuraminidase, endoglycosidase F (endo F), N-glycosidase F (PNGase F), and O-glycanase treatment. Only N-glycosidase F and neuraminidase increased the electrophoretic mobility of the radiolabeled receptor bands, whereas all other glycosidases did not. PNGase F treatment of both radiolabeled receptor bands electroeluted from gel slices reduced the apparent molecular mass of by 16-17 kDa units, that is M(r) = 96,000 to 79,000 and M(r) = 60,000 to 44,000, indicating removal of N-linked oligosaccharide chains of similar size from both species. Neuraminidase treatment caused slight increases in the electrophoretic mobilities suggesting the presence of terminal sialic residues. It is concluded that the Y2 binding proteins are N-linked complex (sialo)glycoproteins with a minimal core protein size of M(r) = 44,000. Furthermore, based on this sensitivity pattern of the glycosidases, the Asn-linked carbohydrate may be of the tri- or tetra-antennary complex type containing terminal sialic acid residues.     

Solubilization of active and stable receptors for vasoactive intestinal peptide from rat liver

Vasoactive intestinal peptide (VIP) receptors were solubilized from rat liver using the zwitterionic detergent CHAPS. Optimal conditions of solubilization were obtained with 5 mM CHAPS and 2.5 mg protein/ml. The binding of 125I-VIP to CHAPS extracts was time- and pH-dependent, saturable and reversible. The following order of potency of unlabeled VIP-related peptides for inhibiting 125I-VIP binding was observed: VIP greater than helodermin greater than peptide histidine isoleucine amide (PHI) greater than rat growth hormone releasing factor (rGRF) greater than secretin. This peptide specificity is identical to that of rat liver membrane-bound receptors. VIP binding activity in the CHAPS extract was destroyed by trypsin or dithiothreitol in accordance with the known sensitivity of membrane-bound receptors to these agents. VIP receptors in CHAPS extracts were stable for at least 5 days at 4 degrees C. Scatchard analysis of equilibrium binding data indicated the presence in CHAPS extracts of high (H) and low (L) affinity binding sites with the following characteristics: KdH = 0.27 nM and BmH = 34 fmol/mg protein; KdL = 51 nM and BmL = 1078 fmol/mg protein. The guanine nucleotide GTP inhibited 125I-VIP binding to soluble receptors and enhanced the dissociation of soluble VIP-receptor complexes, suggesting that GTP-binding proteins were functionally associated with VIP receptors in solution. Gel filtration of solubilized VIP receptors on Sephacryl S-300 revealed a single binding component with a Stokes radius of 6.1 nm. It is concluded that active VIP receptors can be extracted from liver membranes by CHAPS. The availability of this CHAPS-soluble, stable and functional receptor from a tissue which can be obtained in large amounts represents a major step toward the purification of VIP receptors.     

Solubilization of rat lung vasoactive intestinal peptide receptors in the active state. Characterization of the binding properties and comparison with membrane-bound receptors

We demonstrate here that rat lung membrane vasoactive intestinal peptide (VIP) receptors can be extracted in the active state using digitonin. Sepharose 4B gel filtration chromatography was utilized to demonstrate the formation of specific binding complexes between 125I-VIP and solubilized receptors. A rapid soluble receptor assay was established to separate 125I-VIP-receptor complexes from free 125I-VIP, which entailed differential precipitation of the 125I-VIP-receptor complex with polyethylene glycol and bovine gamma-globulin. Using this assay, several detergents were tested for their suitability to extract active VIP receptors, and most favorable results were obtained with digitonin, as judged by specific binding of 125I-VIP to the solubilized receptors. Time course studies indicated that the binding of 125I-VIP to digitonin extract was more rapid than to rat lung membranes. Scatchard analyses of competitive binding data indicated the presence of two classes of binding sites in the digitonin extract, as in the membrane. The values for the dissociation constants (Kd) were 200 pM for Class I and 8 nM for Class II receptors while the values for binding capacity (Bmax) were 200 and 2300 fmol/mg for Class I and II sites, respectively. Although the binding parameters of the two classes were similar to those in the membrane, the pharmacological properties were different, as evidenced by the inability of rat growth hormone releasing factor, a potent VIP agonist in the membrane, to displace specifically bound 125I-VIP from solubilized receptors. The ability to solubilize active VIP receptors represents an important step toward purification of the functional protein.     

Solubilization and hydrodynamic properties of pig atrial muscarinic acetylcholine receptor in dodecyl beta-D-maltoside.

The pig atrial muscarinic acetylcholine receptor (mAcChR) has been solubilized from the membrane-bound state in high yield and in stable conformation by the non-ionic detergent dodecyl beta-D-maltoside (DBM). The yield and selectivity for receptor solubilization is dependent on the detergent/protein ratio during extraction. Extraction at 2 mg of DBM/mg of protein gave a 75% yield of solubilized receptor with a 1.5-fold enrichment. A double-extraction procedure, in which non-receptor protein was first extracted at 0.4 mg of DBM/mg of protein and mAcChR was selectively solubilized by a second extraction at 0.35 mg of DBM/mg of protein, gave a 50% overall yield and a 2.8-fold enrichment. Both preparations had a half-life of about 20 days on ice without addition of muscarinic ligands. Receptor stability was decreased by the presence of cations, particularly bivalent cations, and enhanced by the agonist carbachol. Dissociation constants for the interaction of the DBM-solubilized receptor with the antagonist L-quinuclidinyl benzilate (Kd = 223 pM) and the agonist carbachol (Kd = 100 microM) were similar to those for the digitonin/cholate-solubilized receptor. Pig atrial mAcChR purified in digitonin/cholate and exchanged into DBM displayed reliable hydrodynamic behaviour during sucrose density sedimentation in gradients of 2H2O and H2O and during gel filtration in Sephacryl S-300. DBM is thus the first detergent which will solubilize a stable form of the ligand-free mAcChR in yields similar to those with digitonin, and is the only stabilizing detergent thus far suitable for hydrodynamic studies. DBM is also likely to be similarly useful in studying other membrane proteins for which digitonin has been the solubilizing detergent of choice.     

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.     

Characterization of hepatic lactogen receptor. Subunit composition and hydrodynamic properties

The structure of the membrane-bound and Triton X-100-solubilized female rat liver prolactin receptor has been studied by affinity cross-linking/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and sucrose/H2O and sucrose/D2O density gradient centrifugation. Hydrodynamic characterization revealed that the 125I-human growth hormone receptor-detergent complex represents a molecular species with a Stokes radius of 61 A, a sedimentation coefficient of 5.0 s, and a calculated molecular weight of 158,000. The molecular weight of the receptor was calculated to be 92,000. Three lactogenic hormone-binding species with Mr values of 87,000, 40,000, and 35,000, respectively, were repeatedly found when detergent-solubilized preparations were analyzed using an affinity cross-linking technique. Estrogen treatment of female rats increased the intensity of these bands. Occasionally, an Mr 165,000 hormone-binding species was also found. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis studies (first dimension, nonreducing; second dimension, reducing) demonstrated that disulfide- and nondisulfide-linked hormone-binding species with Mr values of 40,000 and 35,000 are contained within the Mr 87,000 species. It is concluded that the Triton X-100-solubilized female rat liver prolactin receptor has a molecular weight of about 90,000. This molecular species contains Mr 40,000 and Mr 35,000 hormone-binding subunits. It cannot be determined whether these subunits are combined with each other or with as yet undetected subunit(s) to make up the Mr 90,000 species, or whether each one of these subunits is a proteolytic fragment of the Mr 90,000 species.     

Somatostatin receptor subtype-5 mediates inhibition of peptide YY secretion from rat intestinal cultures

Somatostatin-14 (S-14) and somatostatin-28 (S-28) bind to five distinct membrane receptors (SSTRs), but S-28 has higher affinity for SSTR-5. Whether S-28 acting through SSTR-5 regulates inhibition of peptide YY (PYY) secretion was tested in fetal rat intestinal cell cultures. S-28 and S-14 caused dose-dependent inhibition of PYY secretion stimulated by gastrin-releasing peptide, but S-28 was more potent than S-14 (EC(50) 0.04 vs. 13.2 nM). PYY was inhibited by two analogs with affinity for SSTR-5, BIM-23268 and BIM-23052, more potently than S-14 and as effectively as S-28. The SSTR-5 analog L-362855 suppressed PYY equivalent only to S-14, but the structurally related peptide L-372588 (Phe to Tyr at position 2) was equipotent to S-28, whereas L-372587 (Phe to Tyr at position 7) caused no inhibition. An SSTR-2 analog decreased PYY secretion similar to S-14, and an SSTR-3 analog was ineffective. PYY secretion stimulated by phorbol 12-myristate 13-acetate and by forskolin was also more potently suppressed by S-28 and the octapeptide SSTR-5 analogs. The results indicate that S-28 mediates inhibition of gastrin-releasing peptide-stimulated PYY secretion through activation of SSTR-5 and includes suppression of cAMP- and protein kinase C-dependent pathways. Substitution of a single hydroxyl group confers differences in SSTR-5 agonist properties, suggesting region specificity for the intrinsic activity of this receptor subtype.     

Solubilization and hydrodynamic characteristics of the erythropoietin receptor. Evidence for a multimeric complex

In order to study the erythropoietin receptor in its native state, we solubilized erythropoietin-receptor complexes from spleen cell membranes of mice infected with the anemia strain of Friend virus using mild detergents. Among 11 tested detergents, Triton X-100 and Lubrol PX were the most effective. Triton X-100 was therefore selected for this study. The solubilized complexes appeared to be well representative of the total membrane receptor population as indicated by cross-linking experiments and affinity measurements. The hydrodynamic characteristics of the complexes were determined by gel filtration chromatography and ultracentrifugation through sucrose gradients prepared with H2O or D2O. Although erythropoietin-receptor-detergent complexes exhibited some heterogeneity, we determined the following minimal hydrodynamic values: sedimentation coefficient (s20,w): 11.7 +/- 0.8 S, Stokes radius: 7.7 +/- 0.2 nm, partial specific volume: 0.774 +/- 0.017 ml/g, giving a molecular mass of 458 +/- 66 kDa. The contribution of the detergent was estimated to be 28% from the measured partial specific volume, giving an estimated molecular mass of 330 +/- 48 kDa for the erythropoietin-receptor complex. The minimal molecular mass value was significantly greater than those obtained by polyacrylamide gel electrophoresis under denaturing conditions, strongly suggesting that the erythropoietin receptors were present as multimeric complexes. The nature of these complexes is discussed. Beside this major component our results revealed the presence of higher-molecular-mass erythropoietin binding components. We also demonstrated that erythropoietin-receptor complexes could be precipitated with anti-erythropoietin antibodies. This property should greatly improve the purification of erythropoietin receptors.     

Solubilization and various properties of NAD-binding receptor protein from rat brain synaptic membranes

The NAD-binding receptor protein has been solubilized from the synaptic membranes of the rat brain by different detergents. Digitanin proved to be the most effective detergent which exerted no action on the specific binding of [14C]NAD with the solubilized receptor protein. Kinetic parameters of the soluble ligand-receptor complex were studied. The affinity of the solubilized receptor protein to NAD did not change as compared to the protein of native membranes. The specific binding of [14C]NAD was saturated at Kd = 0.53 microM, Bmax = 0.011 nmol per 1 mg of protein. The molecular weight of the soluble NAD-receptor complex determined under native conditions was equal to 115 kDa.     

Solubilization and molecular characterization of active galanin receptors from rat brain.

Galanin receptors were solubilized from rat brain using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). Binding of 125I-galanin to the soluble fraction was time- and temperature-dependent, saturable, and reversible. Scatchard analysis of binding data indicated that the soluble extract contained a single class of galanin binding sites with a Kd of 0.8 nM and a Bmax of 26 fmol/mg of protein. Unlabeled galanin and its fragments galanin(2-29) and galanin(1-15) antagonized the binding of 125I-galanin to CHAPS-solubilized extracts with relative potencies similar to those observed with membrane receptors. Galanin(3-29) was found inactive. Binding of 125I-galanin to CHAPS extracts was inhibited by guanine nucleotides with the following rank order of potency: GMP-P-(NH)P greater than GTP greater than GDP. Molecular analysis of the soluble galanin receptor by covalent cross-linking of 125I-galanin to CHAPS extracts using disuccinimidyl tartrate and further identification on SDS-PAGE indicated that the soluble galanin binding site behaves as a protein of Mr 54,000. After incubation of CHAPS extracts with 125I-galanin, gel filtration on Sephacryl S-300 followed by ultracentrifugation on sucrose density gradient revealed a binding component with the following hydrodynamic parameters: Stokes radius, 5 nm; s20,w, 4.5 S; Mr, 98,000; frictional ratio, 1.6. GMP-P(NH)P treatment of CHAPS extracts gave rise to a molecular form with the following characteristics: Stokes radius, 4 nm; s20,w, 3.3 S; Mr, 57,000; frictional ratio, 1.4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization and assay of phospholipase A2 activity from rat jejunal brush-border membranes

The phospholipase activity of rat jejunal brush-border membranes was examined in the presence of several solubilizing agents, by measuring the hydrolysis of endogenous membrane phospholipids, as well as the hydrolysis of exogenous, radiolabelled substrates. Enzyme activity was highly stimulated by dispersion in 1% solutions of bile salts, or in a synthetic, bile-salt derivative, 3-[(3-cholamidopropyl)dimethylammonio]propanesulphonate (CHAPS). Under these conditions the endogenous membrane phospholipids were largely degraded to free fatty acids and water-soluble phosphate. In the presence of 1% CHAPS, hydrolysis of exogenous phosphatidylcholine was shown to be due to an initial phospholipase A2-type attack followed by a subsequent lysophospholipase-type attack. These activities co-purified with the brush-border membrane. Maximal phospholipase A2 hydrolysis occurred at an alkaline pH of 8-11, with bile-salt detergents present at greater than their critical micellar concentrations. Hydrolysis was completely divalent-ion independent. Phospholipase A2 activity was not stimulated by 50% diethyl ether or ethanol, or in the presence of 1% solutions of Triton X-100, Zwittergent 3-12, sodium dodecyl sulphate, or n-octylglucoside. Stimulation of phospholipase activity by detergents was not related to their effectiveness at solubilizing the membrane proteins. When assayed individually phosphatidylcholine and lysophosphatidylcholine were each hydrolyzed (at the sn-2 and sn-1 positions, respectively) at a rate of approximately 125 nmol/mg protein per min. When assayed together, the two substrates appeared to compete for the same active site over a wide range of concentrations. It was concluded that the brush-border membrane contains an integral membrane protein with phospholipase A2 and lysophospholipase activities, which is specifically stimulated by bile salts and bile salt-like detergents.     

Solubilization of somatostatin receptors in hamster pancreatic beta cells. Characterization as a glycoprotein interacting with a GTP-binding protein

Somatostatin receptors of plasma membranes from beta cells of hamster insulinoma were covalently labelled with 125I-[Leu8,D-Trp22,Tyr25]somatostatin-28 (125I-somatostatin-28) and solubilized with the non-denaturing detergent Triton X-100. Analysis by SDS/PAGE and autoradiography revealed three specific 125I-somatostatin-28 receptor complexes with similar molecular masses (228 kDa, 128 kDa and 45 kDa) to those previously identified [Cotroneo, P., Marie, J.-C. & Rosselin, G. (1988) Eur. J. Biochem. 174, 219-224]. The major labelled complex (128 kDa) was adsorbed to a wheat-germ-agglutinin agarose column and eluted by N-acetylglucosamine. Also, the binding of 125I-somatostatin-28 to plasma membranes was specifically inhibited by the GTP analog, guanosine-5'-O-(3-thiotriphosphate) (GTP[S]) in a dose-dependent manner. Furthermore, when somatostatin-28 receptors were solubilized by Triton X-100 as a reversible complex with 125I-somatostatin-28, GTP[S] specifically dissociated the bound ligand to a larger extent from the soluble receptors than from the plasma-membrane-embedded receptors, the radioactivity remaining bound after 15 min at 37 degrees C being 30% and 83% respectively. After pertussis-toxin-induced [32P]ADP-ribosylation of pancreatic membranes, a 41-kDa [32P]ADP-ribose-labelled inhibitory guanine nucleotide binding protein coeluted with the 128-kDa and 45-kDa receptor complexes. The labelling of both receptor proteins was sensitive to GTP[S]. The labelling of the 228-kDa band was inconsistent. These results support the conclusion that beta cell somatostatin receptors can be solubilized as proteins of 128 kDa and 45 kDa. The major labeled species corresponds to the 128-kDa band and is a glycoprotein. The pancreatic membrane contains a 41-kDa GTP-binding protein that can complex with somatostatin receptors.     

Solubilization and characterization of active somatostatin receptors from rat pancreas

Somatostatin receptors were solubilized from rat pancreatic membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid (CHAPS). The binding of an iodinated somatostatin analog [125I-Tyr3]SMS to the soluble fraction was time-dependent, saturable, and reversible. Scatchard analysis of equilibrium binding data indicated that the soluble extract contained a single class of somatostatin binding sites with a Kd of 0.3 nM and a Bmax of 210 fmol/mg. As observed with membrane-bound receptors, soluble binding receptors were sensitive to the GTP analog GTP gamma S indicating that they are functionally linked to a G protein. A molecular weight of about 400,000 was determined for soluble receptors under native conditions by gel filtration. In denaturing gel electrophoresis, photoaffinity labeling of soluble receptors identified a major protein of Mr = 100,000 and two minor proteins of Mr = 56,000 and 21,000. Isoelectric focusing of soluble receptors revealed that the somatostatin receptor is an acidic protein with pI 4.8. The soluble somatostatin receptor is a glycoprotein which can be specifically bound to the wheat germ agglutinin lectin and eluted by triacetyl-chitotriose.     

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 and hydrodynamic characterization of guanine nucleotide sensitive vasoactive intestinal peptide-receptor complexes from rat intestine

The purpose of this work was to solubilize vasoactive intestinal peptide (VIP) receptors from rat small intestinal plasma membranes and to analyze the nature and function of its molecular form(s) in a nondenaturing environment. Membranes were incubated with 3 nM 125I-VIP, washed, and treated with 1% Triton X-100. Chromatography on Sephadex G-50 showed that 60% of the extractable radioactivity was eluted with macromolecular components in the void volume. This radioactive material was dramatically reduced when 1 microM unlabeled VIP was present in the incubation medium or when membranes were pretreated with trypsin or dithiothreitol. Macromolecular components that had bound 125I-VIP were further chromatographed on Sephacryl S-300. Two peaks were observed: a major one (80%) and a minor one (20%) with Stokes radii of 5.2 and 3.1 nm, respectively. The labeling of both components was inhibited by unlabeled VIP or peptide with NH2-terminal histidine and COOH-terminal isoleucine amide (a VIP agonist). The presence of GTP (0.1 mM) in the incubation medium of membranes completely abolished the labeling of the 5.2-nm component but did not affect that of the 3.1-nm one. Moreover, GTP induced dissociation of 125I-VIP from the 5.2-nm component isolated by Sephacryl S-300 chromatography. This effect was time dependent and nucleotide specific. In contrast, GTP did not affect the stability of the 3.1-nm component. After cholera toxin catalyzed [32P]ADP-ribosylation of membranes, chromatography of solubilized material on Sephacryl S-300 showed that a peak of 32P radioactivity was coeluted with the 5.2-nm component.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization of the vasopressin receptor from rat liver plasma membranes. Evidence for a receptor X GTP-binding protein complex

The GTPase activity of plasma membranes isolated from rat livers was stimulated 20% over basal by vasopressin. A concentration dependency curve showed that maximal stimulation was obtained with 10(-8) M vasopressin. The vasopressin-stimulated GTPase activity was not inhibited in plasma membranes that had been ADP-ribosylated with either cholera toxin or pertussis toxin. Identical results were obtained from plasma membranes that had been solubilized with 1% digitonin. When membranes that had been solubilized after preincubation with [3H]vasopressin were subjected to sucrose gradient centrifugation, the majority of protein-bound [3H]vasopressin migrated as a single band with a sedimentation constant of 16.8 S. Moreover, there was a GTPase activity that migrated with the bound [3H]vasopressin. This peak of bound [3H]vasopressin was decreased by 90% when the sucrose gradient centrifugation was run in the presence of 10 microM guanosine 5'-O-(thiotriphosphate). When the 16.8 S peak of bound [3H]vasopressin was further purified over a wheat germ lectin-Sepharose column, a GTPase activity co-eluted from the column with the protein-bound [3H]vasopressin. Direct evidence that a GTP-binding protein was present in the 16.8 S peak was obtained by the immunodetection of a 35-kDa beta subunit of a GTP-binding protein. These results support the conclusion that liver plasma membranes contain a GTP-binding protein that can complex with the vasopressin receptor.     

Characterization and photoaffinity labeling of a calcitonin gene-related peptide receptor solubilized from human cerebellum.

Calcitonin gene-related peptide (CGRP) receptors were solubilized from human (h) cerebellum with use of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). Scatchard analysis of equilibrium binding data indicated that the soluble extract contained a single class of CGRP binding sites with apparent dissociation constants of 50 pM for the intact 125I-hCGRP-I(1-37) and 160 pM for the antagonist 125I-hCGRP-I(8-37). Unlabeled hCGRP-I and -II and hCGRP-I(8-37) displaced 125I-hCGRP-I from solubilized CGRP receptors with similar potencies (ID50 = 70-150 pM). Human CGRP-I(15-37), -(21-37), and -(28-37) were less potent (ID50 greater than or equal to 70 nM), suggesting that amino acid residues 8-14 may be important for maintaining high binding affinity. A novel photoreactive analogue of hCGRP-I, 125I-[C gamma-(4-azidoanilino)Asp3] hCGRP-I, was prepared by carbodiimide coupling of 4-azidoaniline to 125I-hCGRP-I. Photoaffinity labeling of soluble CGRP receptors with the photoreactive analogue and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed three specifically labeled binding proteins with apparent molecular weights (Mr) of 60,000, 54,000, and 17,000. Cross-linking of 125I-hCGRP-I and -II and 125I-hCGRP-I(8-37) to soluble CGRP binding sites using disuccinimidyl suberate revealed three specifically labeled binding proteins with the same Mr. The C-terminal fragment 125I-hCGRP-I(8-37), unlike the intact peptide, was, furthermore, cross-linked specifically to a 95,000 Mr protein. The CGRP receptor is N-glycosylated. Treatment with endoglycosidase F/N-glycosidase F converted the 60,000 and 54,000 to 46,000 and 41,000 Mr components.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analogs of pancreatic polypeptide and peptide YY with a locked PP-fold structure are biologically active

Pancreatic polypeptide (PP), peptide YY (PYY) and neuropeptide Y (NPY), members of the PP-fold family share a high degree of sequence homology. Nuclear magnetic resonance (NMR) and X-ray crystallography studies have shown these peptides can adopt a tightly organized tertiary structure called the PP-fold, which has long been assumed to be the active structure of this family of peptides. To date, however, no studies have been completed with PYY and PP which confirm if the PP-fold structure is important for their physiological actions. The aim of the study was to test if PYY and PP locked into the PP-fold maintained biological activity. Therefore, we designed and produced analogs of PP and PYY in a cyclic conformation with two cysteine amino acid substitutions at the N-terminus and at position 27. These were oxidized to form a cysteine disulfide bond locking the peptides into the PP-fold structure. Studies demonstrate that the cyclic analogs have both similar in vivo activity to their parent molecules, and affinity for the Y2 and Y4 receptors. Results suggest that the proposed PP and PYY-fold is likely to be their biologically active conformation.