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PubMed1997

The type V transforming growth factor beta receptor is the putative insulin-like growth factor-binding protein 3 receptor.

Insulin-like growth factor-binding protein 3 (IGFBP-3) has been shown to inhibit cell growth by IGF-dependent and -independent mechanisms. The putative cell-surface IGFBP-3 receptor that mediates the IGF-independent growth inhibition has not been identified. Here we show that recombinant human IGFBP-3 inhibits 125I-transforming growth factor (TGF)-beta1 binding to the type V TGF-beta receptor (Mr 400,000) in mink lung epithelial cells. We also demonstrate that the approximately 400-kDa 125I-IGFBP-3 affinity-labeled putative IGFBP-3 receptor is immunoprecipitated by specific antiserum to the type V TGF-beta receptor. The 125I-IGFBP-3 affinity labeling of the putative receptor and IGFBP-3-induced growth inhibition as measured by DNA synthesis in these cells is blocked by a TGF-beta1 peptide antagonist. The 125I-IGFBP-3 affinity-labeled putative receptor can only be detected in cells expressing the type V TGF-beta receptor, but not in cells lacking the type V TGF-beta receptor. These results indicate that the type V TGF-beta receptor is the putative IGFBP-3 receptor and that IGFBP-3 is a functional ligand for the type V TGF-beta receptor.

Animals↗
PubMed1983

Properties of the active site lysyl residue of mitochondrial aspartate aminotransferase in solution.

Two vitamin B6 derivatives, N-bromoacetylpyridoxamine (BAPM) and its phosphate ester have been found to be affinity-labeling reagents for mitochondrial aspartate aminotransferase (EC 2.6.1.1). These derivatives were first shown to react with a critical sulfhydryl group in tryptophan synthase (Higgins, W., and Miles, E. W. (1978) J. Biol. Chem. 253, 4648-4652). In the apoaminotransferase, BAPM has now been found to inactivate by covalently modifying a critical lysyl residue, preventing reconstitution of the apoenzyme by pyridoxal 5'-phosphate. The dependence of the rate of inactivation upon the concentration of the reagent is consistent with a rapid equilibrium binary complex formation prior to the inactivation reaction. Both the dissociation constant for this complex and the rate of the reaction leading to inactivation are dependent on pH. BAPM binds best from pH 7.5 to 8.5. The rate of inactivation increases from pH 6 to 9. Succinate and phosphate competitively bind to the apoenzyme, protecting against BAPM inactivation. The C-5'-phosphorylated derivative is rapidly and tightly bound by the apotransaminase to form an inactive, noncovalent adduct. This bound reagent subsequently alkylates Lys-258. The rate of this covalent incorporation increases from pH 6 to 9 and is greater than the rate of BAPM modification at all pH values. The effect of pH on the reaction rates of both pyridoxal derivatives is interpreted to indicate protonation of Lys-258 at neutral pH values. These derivatives may also be analogs to a reaction intermediate different from those observed in other affinity-labeling studies. The ionization states of the Lys-258 epsilon-amino group apparently vary with the nature of the affinity label. These variations can be explained in terms of changing ionization states of Lys-258 in the steps of catalysis as well as in terms of the occupancy of charged sites on the protein by active site-directed substrates or inhibitory compounds.

Affinity Labels↗
PubMed1983

Parallel decreases in the expression of receptors for insulin and insulin-like growth factor I in a mutant human fibroblast line.

The receptors for insulin and the insulin-like growth factor (IGF) I are two structurally homologous disulfide-linked multisubunit complexes of apparent Mr = 350,000. The similar subunit structures of these two types of receptors suggested that their genetic expression might be affected by common genetic defects. We have examined this possibility in an insulin-resistant, diabetic patient who exhibits decreased insulin binding activity. The receptors for IGF-I and insulin in skin fibroblasts from this patient were affinity labeled with 125I-IGF-I and 125I-insulin, respectively, and visualized by electrophoresis and autoradiography in polyacrylamide gels. Control fibroblasts exhibited the usual affinity labeling of the disulfide-linked Mr = 350,000 insulin and IGF-I receptor structures. The intensity of labeling of both receptor types in the patient's fibroblasts was less than in control fibroblasts. Binding data indicated that this decrease is due to a decreased receptor number with little or no decrease in affinity for the respective ligands. The high-affinity IGF-II receptor in fibroblasts affinity labeled with 125I-IGF-II or 125I-IGF-I consists of a single polypeptide not disulfide linked to any other membrane component. The molecular size and intensity of labeling of the IGF-II receptor in the patient's fibroblasts were unaltered when compared with those of controls. These observations suggest that a common genetic defect alters the expression of the homologous receptor structures for insulin and IGF-I.

Cell Line↗
PubMed1991

Characterization of a novel receptor in toad retina with dual specificity for insulin and insulin-like growth factor I.

The biochemical properties of insulin receptors from toad retinal membranes were examined in an effort to gain insight into the role this receptor plays in the retina. Competition binding assays revealed that toad retinal membranes contained binding sites that displayed an equal affinity for insulin and insulin-like growth factor I (IGF-I). Affinity labeling of toad retinal membrane proteins with 125I-insulin resulted in the specific labeling of insulin receptor alpha-subunits of approximately 105 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partially reduced (alpha beta-heterodimer) receptors affinity-labeled with 125I-insulin indicated the presence of a disulfide-linked beta-subunit of approximately 95 kDa. Endoglycosidase F digestion of the affinity-labeled alpha-subunits increased their mobility by reducing their apparent mass to approximately 83 kDa. This receptor was not detected by immunoblot analysis with a site-specific antipeptide antibody directed against residues 657-670 of the carboxy terminal of the human insulin receptor alpha-subunit, whereas this antibody did label insulin receptor alpha-subunits from pig, cow, rabbit, and chick retinas. In in vitro autophosphorylation assays insulin stimulated the tyrosine phosphorylation of toad retina insulin receptor beta-subunits. These data indicate that toad retinal insulin receptors have a heterotetrameric structure whose alpha-subunits are smaller than other previously reported neuronal insulin receptors. They further suggest that a single receptor may account for both the insulin and IGF-I binding activities associated with toad retinal membranes.

Animals↗
PubMed1974

Chemical conversion of aspartic acid 52, a catalytic residue in hen egg-white lysozyme, to homoserine.

Hen egg-white lysozyme (EC 3.2.1.17) was specifically esterified at aspartic acid 52 by the affinity labeling reagent 2',3'-epoxypropyl beta-glycoside of di-(N-acetyl-D-glucosamine) [Eshdat et al. (1973) J. Biol. Chem.248, 5892]. The disulfide bonds of the affinity-labeled enzyme and the aspartic acid 52-ester bond were reduced with dithiothreitol and sodium borohydride, respectively, resulting in the removal of the affinity label. The reduced protein contained 0.9 mole of homoserine and 1 mole less of aspartic acid per mole of protein, as compared to the native enzyme. It was reoxidized by a mixture of reduced and oxidized glutathione to yield a modified protein that possessed one-tenth of the activity of native lysozyme (presumably due to a contamination by regenerated lysozyme formed as a result of hydrolysis of the aspartic acid 52-ester bond during the chemical treatment). The native enzyme, after reduction and reoxidation in the same manner, retained its amino-acid composition, full enzymatic activity, and fluorescence properties. The modified lysozyme, containing homoserine 52, showed the same fluorescence spectrum as the native enzyme. With both proteins, the fluorescence maximum shifted to the blue to a similar extent upon the addition of the saccharide inhibitors tri-(N-acetyl-D-glucosamine) and the cell-wall tetrasaccharide (GlcNAc-MurNAc)(2). The modified enzyme bound these two saccharides with nearly the same binding constants as those found for native lysozyme and for lysozyme that was reduced and reoxidized. Since the side chain of homoserine is similar in size to that of aspartic acid, it is concluded that the loss of enzymatic activity is the direct result of the chemical modification of the carboxyl side chain of aspartic acid 52, thus showing that this amino acid is essential for the catalytic action of the enzyme.

Amino Acid Sequence↗
PubMed1989

The intermediate-affinity interleukin (IL)2 receptor expressed on Theileria annulata-infected cells comprises a single IL 2-binding protein. Partial characterization of bovine IL2 receptors.

Bovine high-, intermediate- and low-affinity interleukin 2 receptors (IL2R) were studied by ligand binding and affinity labeling using 125I-labeled IL2 and homobifunctional chemical cross-linking reagents. High- (Kd = 17 pM) and low-affinity (Kd greater than 6 nM) IL 2R were detected on concanavalin A-activated peripheral blood lymphocytes (PBL). Theileria annulata (TA)-infected autonomously growing PBL (TA-PBL) express predominantly intermediate-affinity IL2R (Kd = 1 nM). Affinity-labeling studies revealed that the high-affinity IL2R comprises a 55-kDa (L chain) and an additional 90-kDa IL 2-binding protein (H chain). TA-PBL express predominantly the H chain. In contrast to the human chain, the bovine form was not separable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/urea into the two distinct bands H1 and H2 and ran in parallel to the human H2 band. These results suggest (a) that the bovine intermediate-affinity IL2R comprises a single H chain and (b) that the single H chain and the L chain are sufficient to form the functional high-affinity bovine IL2R.

Affinity Labels↗
PubMed1994

Activin-A binds to a heterotrimeric receptor complex on the vascular endothelial cell surface. Evidence for a type 3 activin receptor.

The effect of transfection of the type 2 activin receptor, ACTR2, on binding of 125I-activin-A to the surface of bovine aortic endothelial cells (BAEC) was investigated. BAEC transfected either with full-length ACTR2 or with a truncated form of ACTR2 lacking the intracellular kinase domain (ACTR2T) displayed two classes of 125I-activin-A binding sites, one of high affinity (Kd = 250-254 pM) and one of low affinity (Kd = 6.5-16 nM). Affinity labeling of ACTR2-transfected BAEC with 125I-activin-A revealed labeled species of 55, 95, 100, and 160 kDa, all four of which were immunoprecipitated by an anti-ACTR2 monoclonal antibody. Only the 95- and 100-kDa species, however, were immunoprecipitated following denaturation of the affinity-labeled cell lysate with SDS. BAEC transfected with an epitope-tagged form of ACTR2T (ACTR2TMyc) displayed intense 55- and 70-kDa affinity-labeled forms of the truncated receptor, together with a 160-kDa species. As with the full-length receptor, the 160-kDa species associated non-covalently with ACTR2TMyc. These data indicate that, in vascular endothelial cells, ACTR2 forms a high affinity heterotrimeric receptor complex with activin-binding proteins characteristic of type 1 and type 3 activin receptors, and that formation of the complex does not require the kinase domain of ACTR2.

Activin Receptors↗
PubMed1977

Affinity alkylation labels two subunits of the reduced acetylcholine receptor from mammalian muscle.

The acetylcholine receptor from denervated rat skeletal muscle was purified by affinity chromatography and, after reduction, was treated with the affinity alkylating agent 4-(N-maleimido)benzyltri[3H]methylammonium iodide. The receptor specifically incorporated approximately 1 mol of alkylating agent per mol of 125I-labeled alpha-bungarotoxin bound. Analysis of the labeled receptor by polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that two subunits were labeled; their apparent molecular weights were 45,000 and 49,000. These results suggest that the affinity reagent labels a second site for acetylcholine binding in the muscle receptor that is not labeled in receptors from Electrophorus or Torpedo.

Acetylcholine↗
PubMed1989

Functional atrial natriuretic peptide receptor in human adrenal tumor.

The effects of synthetic human atrial natriuretic peptide (ANP) on the release of catecholamines, aldosterone, or cortisol were observed in human adrenal tumors obtained surgically from patients with pheochromocytoma, primary aldosteronism, or Cushing's syndrome, respectively. Each tumor tissue or adjacent normal cortical tissue was sectioned into slices, which were incubated in medium-199 in the presence or absence of adrenocorticotrophin (ACTH) and ANP. The amounts of epinephrine, norepinephrine, aldosterone, or cortisol released into the medium were measured. Existence of ANP receptors on the adrenal tissues was examined by binding assays, affinity labeling, and immunohistochemistry. Release of catecholamines from pheochromocytoma tissues was inhibited by ANP, and the presence of the ANP receptor on pheochromocytoma was further demonstrated by both binding assays and affinity labeling; Scatchard analysis revealed a single class of binding sites for ANP with a Kd of 1.0 nM and a Bmax of 0.4 pmol/mg of protein and the molecular size was estimated as 140 and a 70 kDa under nonreducing and reducing conditions, respectively. The presence of ANP receptors in pheochromocytoma was demonstrated by immunohistochemistry. ANP inhibited both basal and ACTH-stimulated aldosterone secretion in the slices of normal cortex, and localization of ANP receptors in zona glomerulosa cells was also demonstrated. However, ANP did not inhibit basal and ACTH-stimulated aldosterone and cortisol secretion in both tissue slices from aldosteronoma and Cushing's adenoma. Consistent with these observations, the absence of ANP receptors in adenoma tissues was determined by binding assays, affinity labeling, and immunohistochemistry.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex↗
PubMed1988

Partial purification of androgen receptor from hypertrophic human prostate.

For purification of androgen receptor from hypertrophic human prostate, solutions used for elution of androgen receptor from DNA Sepharose, affinity labeling of the receptor and ability of affinity gel to retain the receptor were examined. Elution with 20 mM pyridoxal 5'-phosphate of the receptor from DNA Sepharose was more efficient than that with diluted pyridoxal 5'-phosphate, high ionic solution or various concentrations of Mg++, 3H-dihydrotestosterone bromoacetate was applicable to covalent binding with partially purified androgen receptor regardless of the low specificity of the ligand. Affinity gel of thiopropyl-Sepharose 6B coupled to 17 alpha-(2', 3'-epoxy-propyl)-5 alpha-dihydrotestosterone was better than Affigel 102 coupled to N-[3-(3-oxo-5 alpha-androstane-17 beta-yloxycarbonyl) propionyloxy] succimide or aminoethyl-Sepharose 4B coupled to 17 alpha-carboxyethynyl testosterone with respect to the rate of retention of androgen receptor. In view of these observations, the following purification procedures were constructed: Removal of DNA Sepharose-binders from the cytosol, 40% ammonium sulfate precipitation, affinity chromatography using thiopropyl-Sepharose 6B coupled to 17 alpha-(2',3'-epoxypropyl)-5 alpha-dihydrotestosterone, and DNA Sepharose chromatography. After affinity labeling of the receptor thus obtained, the molecular weight was estimated. Some 1300-fold purification with a yield of 0.25% of the androgen receptor was achieved. The molecular weight of the receptor was mainly 45 K with 90 K in a lesser amount. The Stokes radius was calculated as 30 A.

Chromatography, Affinity↗
PubMed1995

Canine kidney glucagon receptor: evidence for a structurally-different, tissue-specific variant of the glucagon receptor.

125I-Glucagon was directly cross-linked to its receptor sites on the MDCK plasma membranes using a UV irradiation procedure. Analysis of the affinity labeled membranes by SDS-PAGE and autoradiography, demonstrated the presence of a single band at 74 kDa. The incorporation of radiolabeled glucagon into this band was abolished by the presence of excess unlabeled hormones, thus indicating a specificity of labeling. Also this band was observed in affinity labeled dog kidney plasma membranes. The size of the MDCK and the dog kidney glucagon receptors were consistently larger than that of the dog liver receptor as judged by electrophoretic mobility. Treatments with neuraminidase, endoglycosidase F, or N-glycanase failed to convert the renal form into the hepatic form of the receptor. Proteolytic mapping of the MDCK and the dog liver glucagon receptors revealed that major domains of both proteins are remarkably similar, yet transient variations in the size of the fragments could be detected after short duration digestions. Overall the data presents evidence that the dog renal receptor represents a structurally unique isoform of the glucagon receptor.

Animals↗
PubMed2006

Structural basis for macrolactonization by the pikromycin thioesterase.

Polyketides are a class of biologically active microbial and plant-derived metabolites that possess a high degree of structural and functional diversity and include many human therapeutics, among them anti-infective and anti-cancer drugs, growth promoters and anti-parasitic agents. The macrolide antibiotics, characterized by a glycoside-linked macrolactone, constitute an important class of polyketides, including erythromycin and the natural ketolide anti-infective agent pikromycin. Here we describe new mechanistic details of macrolactone ring formation catalyzed by the pikromycin polyketide synthase thioesterase domain from Streptomyces venezuelae. A pentaketide phosphonate mimic of the final pikromycin linear chain-elongation intermediate was synthesized and shown to be an active site affinity label. The crystal structures of the affinity-labeled enzyme and of a 12-membered-ring macrolactone product complex suggest a mechanism for cyclization in which a hydrophilic barrier in the enzyme and structural restraints of the substrate induce a curled conformation to direct macrolactone ring formation.

Binding Sites↗
PubMed1999

Nitric oxide suppresses apoptosis via interrupting caspase activation and mitochondrial dysfunction in cultured hepatocytes.

Nitric oxide (NO) is a potent inhibitor of apoptosis in many cell types, including hepatocytes. We and others have described NO-dependent decreases in caspase activity in cells undergoing apoptosis. However, previous work has not determined whether NO disrupts the proteolytic processing and thus the activation of pro-caspases. Here we report that NO suppresses proteolytic processing and activation of multiple pro-caspases in intact cells, including caspase-3 and caspase-8. We found that both exogenous NO as well as endogenously produced NO via adenoviral inducible NO synthase gene transfer protected hepatocytes from tumor necrosid factor (TNF) alpha plus actinomycin D (TNFalpha/ActD)-induced apoptosis. Affinity labeling with biotin-VAD-fmk of all active caspase species in TNFalpha-mediated apoptosis identified four newly labeled spots (activated caspases) present exclusively in TNFalpha/ActD-treated cells. Both NO and the caspase inhibitor, Ac-DEVD-CHO, prevented the appearance of the four newly labeled spots or active caspases. Immunoanalysis of affinity labeled caspases demonstrated that caspase-3 was the major effector caspase. Western blot analysis also identified the activation of caspase-8 in the TNFalpha/ActD-treated cells, and the activation was suppressed by NO. Furthermore, NO inhibited several other events associated with caspase activation in cells, including release of cytochrome c from mitochondria, decrease in mitochondrial transmembrane potential, and cleavage of poly(ADP-ribose) polymerase in TNFalpha/ActD-treated cells. These findings indicate the involvement of multiple caspases in TNFalpha-mediated apoptosis in hepatocytes and establish the capacity of NO to inhibit not only active caspases but also caspase activation.

Animals↗
PubMed1987

Improved methods for the detection of unique sequences in Southern blots of mammalian DNA by non-radioactive biotinylated DNA hybridization probes.

Biotinylated DNA hybridization probes offers a stable, cheap and non-radioactive alternative to probes labelled with 32P. Insufficient sensitivity has, however, up till now, been prohibitive for the use of such probes in detecting unique sequences in Southern blots of human DNA. By optimizing the steps in the procedure we have improved the sensitivity enough for such use. We have showed (1) that long probes (greater than 500 nucleotides) perform unproportionally better than short probes; (2) that a simple affinity labelling with avidin alkaline phosphatase conjugate performs better than laborious immunochemical systems; (3) that use of 3% BSA as blocking agent at 37 degrees C and the presence of 0.5 mol/l NaCl together with 1% BSA during the affinity labelling nearly eliminate background staining; (4) that a dramatic gain in sensitivity is gained by affinity labelling at pH 9.0 instead of 7.5; (5) that biotin-labelling can be highly reproducibly performed on a preparative scale with cheap and easily synthesized bio-11-dUTP in a two step nick-translation and (6) that biotinylated probes and hybridization mixtures can be stored for months and reused. The study has resulted in the presentation of a fast procedure, which is generally applicable to routine DNA diagnostic work, also in parts of the world where it is difficult to get a regular supply of 32P.

Base Sequence↗
PubMed1996

Integrin-associated protein is a receptor for the C-terminal domain of thrombospondin.

The C-terminal "cell-binding domain" (CBD) of thrombospondin-1 (TS1) is a binding site for many cell types. Cell-binding peptides based on the sequence RFYVVM from the CBD of TS1 affinity label a 52-kDa cell surface glycoprotein, which we show is integrin-associated protein (IAP or CD47). IAP associates with alpha v beta 3 and thereby modulates the activity of several integrins. Cells that express IAP bind strongly to TS1, the CBD, and its active cell-binding peptides while IAP negative cells do not. The 52-kDa protein is affinity labeled on IAP-positive but not IAP-negative cells, and monoclonal antibodies against IAP specifically immunoprecipitate the affinity-labeled 52-kDa protein from lysates of IAP-positive cells. Consistent with the association of IAP with alpha v beta 3 integrin, the labeled 52-kDa protein is immunoprecipitated by an anti-alpha v beta 3 antibody. Endothelial cells exhibit chemotaxis toward TS1 (at concentrations above 10 nM) and RFYVVM peptides. Chemotaxis to both agents is specifically inhibited by a function blocking anti-IAP monoclonal antibody. These data establish IAP (CD47) as a receptor for the CBD of TS1 and suggest a mechanism for the well established effects of the CBD on cell motility.

Amino Acid Sequence↗
PubMed2000

Identification of catalytic nucleophile of Escherichia coli gamma-glutamyltranspeptidase by gamma-monofluorophosphono derivative of glutamic acid: N-terminal thr-391 in small subunit is the nucleophile.

gamma-Glutamyltranspeptidase (EC 2.3.2.2) is the enzyme involved in glutathione metabolism and catalyzes the hydrolysis and transpeptidation of gamma-glutamyl compounds such as glutathione and its derivatives. The reaction is thought to proceed via a gamma-glutamyl-enzyme intermediate where a hitherto unknown catalytic nucleophile is gamma-glutamylated. Neither affinity labeling nor site-directed mutagenesis of conserved amino acids has succeeded so far in identifying the catalytic nucleophile. We describe here the identification of the catalytic nucleophile of Escherichia coli gamma-glutamyltranspeptidase by a novel mechanism-based affinity labeling agent, 2-amino-4-(fluorophosphono)butanoic acid (1), a gamma-phosphonic acid monofluoride derivative of glutamic acid. Compound 1 rapidly inactivated the enzyme in a time-dependent manner (k(on) = 4.83 x 10(4) M(-1) s(-1)). The inactivation rate was decreased by increasing the concentration of the substrate. The inactivated enzyme did not regain its activity after prolonged dialysis, suggesting that 1 served as an active-site-directed affinity label by phosphonylating the putative catalytic nucleophile. Ion-spray mass spectrometric analyses revealed that one molecule of 1 phosphonylated one molecule of the small subunit. LC/MS experiments of the proteolytic digests of the phosphonylated small subunit identified the N-terminal peptide Thr391-Lys399 as the phosphonylation site. Subsequent MS/MS experiments of this peptide revealed that the phosphonylated residue was Thr-391, the N-terminal residue of the small subunit. We conclude that the N-terminal Thr-391 is the catalytic nucleophile of E. coli gamma-glutamyltranspeptidase. This result strongly suggests that gamma-glutamyltranspeptidase is a new member of the N-terminal nucleophile hydrolase family.

Aminobutyrates↗
PubMed1991

Evidence against posttranslational glycosylation of rat glucocorticoid receptors.

The observed M(r) of the rat glucocorticoid receptor on denaturing polyacrylamide gels is 7-11 kDa higher than that deduced from the cloned cDNA sequence of the receptor. Posttranslational modification of the receptor, such as glycosylation, could account for this difference in mol wt. Indeed, several reports have appeared in the literature suggesting that glucocorticoid receptors contain sugar moieties. We have used a variety of methods to determine whether the receptor is glycosylated, i.e., digestion of affinity labeled receptors with various glycosidases, immunoadsorption of receptors after whole cell labeling with [3H]D-glucosamine, and ion exchange and lectin column chromatography of affinity labeled receptors. With each of these methods, there was no evidence for glycosylation of the receptor protein. We therefore conclude that there is no significant amount of either N-linked glycosylation, or O-linked glycosylation with D-glucosamine, of the rat glucocorticoid receptor.

Affinity Labels↗
PubMed1990

[Structural characterization of the somatostatin receptors on rat cerebrocortical membranes].

We characterized structurally the receptors for somatostatin in rat cerebral cortex by affinity labeling with [125I-Tyr1] somatostatin. [125I-Tyr1] somatostatin was cross-linked to cerebrocortical membranes using photoreactive cross-linker: N-5-azido-2-nitrobenzoyloxy-succinimide. Analysis by autoradiography revealed a broad band centered at Mr = 72,000 in the presence or absence of dithiothreitol. Affinity labeling of and specific [125I-Tyr1] somatostatin binding to cerebrocortical membranes were decreased similarly by adding unlabeled somatostatin or nonhydrolyzable guanine nucleotide analogue, guanyl-5'-yl imidodiphosphate, in a dose dependent manner. The pretreatment of cerebrocortical membranes with islet activating protein resulted in a decrease in subsequent affinity labeling of the protein. The cross-linked protein could be solubilized with Zwittergent 3-12 and poorly with digitonin, triton X-100 and NP-40. When exposed to agarose-coupled lectins, the solubilized labeled protein was absorbed to wheat germ agglutinin, partially to ricin communis-II, and not to concanavalin A or lentil lectin. The Mr = 72,000 protein bound to wheat germ agglutinin-agarose was eluted with not only N,N',N"-triacetylchitotriose but also N-acetylglucosamine. These results suggest that somatostatin receptors on cerebrocortical membranes are a monomeric glycoprotein with a Mr = 70,000 containing no disulfide-linked binding subunit, which is coupled to islet activating protein-sensitive guanine nucleotide regulatory protein.

Animals↗