Interaction of hepatic asialoglycoprotein receptor with asialoorosomucoid and galactolyzed lysosomal α-glucosidase

An asialoglycoprotein receptor was isolated from murine liver and purified more than 1600-fold using 2-fold affinity chromatography on asialoorosomucoid-Sepharose. The purified receptor did not interact with ¹²⁵I-orosomucoid, but bound to ¹²⁵I-asialoorosomucoid. The binding of the receptor to asialoorosomucoid was saturable. The dissociation constant of the receptor-asialoorosomucoid complex was 0.4·10⁻⁹ M. The molecular mass of the receptor, as determined with the use of specific antibodies by the immunoblotting method, was 43 kDa. High concentrations of unlabeled asialoorosomucoid and of n-aminophenyl-β-d-galactosyl derivatives of bovine serum albumin, ovalbumin and acid α-glucosidase from human liver inhibited the binding of the receptor to ¹²⁵I-asialoorosomucoid almost completely. The binding of the receptor to ¹²⁵I-galactolyzed α-glucosidase was pH-dependent, with the pH optimum at 8.0–9.0. It was shown that, as in the case of ¹²⁵I-asialoorosomucoid, the binding of the ¹²⁵I-galactosyl derivative of α-glucosidase occurred in the presence of Ca²⁺ and was inhibited by N-acetylgalactosamine. Glycoproteins containing galactose as a terminal residue inhibited the interaction of the receptor with ¹²⁵I-galactolyzed α-glucosidase. The possibility of directed transport of the galactolyzed α-glucosidase derivative into parenchymous liver cells using receptor-mediated endocytosis is discussed.     

Uptake and degradation of 125I-labeled rat asialoorosomucoid by the perfused rat liver

The uptake and degradation of a homologous rat serum asialoglycoprotein, 125I-asialoorosomucoid, and the effects on this metabolism by leupeptin, a proteinase inhibitor, were studied in the perfused rat liver. 125I-Asialoorosomucoid was rapidly taken up by the liver (t1/2 = 5.7 min) and acid-soluble degradation products began to appear in the circulating perfusate medium after 20-30 min. These products accounted for 60-65% of the initially added radioactivity after 90 min of perfusion. The early events in the galactose-mediated uptake of 125I-asialoorosomucoid were unchanged by the presence of leupeptin. However, the appearance of acid-soluble degradation products was greatly reduced when livers had been pretreated with the inhibitor (1.0 mg for 60 min). This effect corresponded with an increase in acid-precipitable material being located within the lysosomal-rich fraction from homogenates of leupeptin-treated livers. Leupeptin inhibited degradation of 125I-asialoorosomucoid by approx. 85% relative to control values over 90 min of perfusion. Inhibition of asialoorosomucoid degradation was also demonstrated in vitro. Leupeptin (1.0 mM) reduced hydrolysis of this glycoprotein substrate by greater than 50% during a 24 h incubation with isolated lysosomal enzymes. The thiol proteinases, cathepsin B, H and L, which are known to be inhibited by leupeptin, are apparently involved in initiating digestion of rat 125I-asialoorosomucoid within liver lysosomes. As a result of inhibition by leupeptin both in the perfused liver and in vitro very limited changes occurred in the native molecular weight of the starting glycoprotein.     

Detection and isolation of a hepatic membrane receptor for ferritin

A ferritin receptor has been detected on isolated rat hepatocytes and has been partially purified from rat liver using affinity chromatography. Isolated hepatocytes exhibit approximately 30,000 ferritin binding sites/cell with a binding association constant (Ka) of 1 x 10(8) mol-1 liter. A binding assay has been developed which utilizes a hepatic ferritin receptor coupled to a microparticulate support to facilitate separation of bound and free ligand. This method yielded a Ka of 3 x 10(8) mol-1 liter for the purified hepatic ferritin receptor. Binding of ferritin to the insolubilized receptor was partially inhibited by human lactoferrin but unaffected by 200-fold molar excess of bovine albumin, rat transferrin, or human asialoorosomucoid.     

The galactose-recognizing system of rat peritoneal macrophages; identification and characterization of the receptor molecule

Resident rat peritoneal macrophages express a galactose-recognizing system, which mediates binding and uptake of cells and glycoproteins exposing terminal galactose residues. Here we describe the identification, isolation, and characterization of the corresponding receptor molecule. Using photoaffinity labelling of adherent peritoneal macrophages with the 4-azido-6-125I-salicylic acid derivative of anti-freeze glycoprotein 8 followed by SDS-PAGE and autoradiography, we identified the receptor of these cells as a protein with an apparent molecular mass of 42 kDa. Furthermore, cell surface receptors were radioiodinated by an affinity-supported labelling technique using the conjugate of asialoorosomucoid and lactoperoxidase, followed by extraction and isolation by affinity chromatography. Finally, the native receptor was isolated and analysed. To estimate its binding activity in solutions, a suitable binding assay was developed, using the precipitation of receptor-ligand complex with polyethylene glycol to separate bound from unbound 125I-asialoorosomucoid, which was used as ligand. It is shown that the isolated receptor binds to galactose-exposing particles and distinguishes between sialidase-treated and -untreated erythrocytes, similar to peritoneal macrophages. The binding characteristics of the membrane-bound and the solubilized receptor are described in the following paper of Lee et al.     

Comparison of the binding of chicken alpha-macroglobulin and ovomacroglobulin to the mammalian alpha 2-macroglobulin receptor

Chicken alpha-macroglobulin (alpha M) and ovomacroglobulin were purified by Ni+2 chelate chromatography. These proteins had similar subunit structure as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chicken alpha M bound 1.0 mol and ovomacroglobulin bound 0.8 mol 125I-trypsin per mol inhibitor, respectively. Ovomacroglobulin cleared rapidly from the circulation of mice, and the clearance was inhibited by asialoorosomucoid, but native chicken alpha M cleared slowly (t 1/2 greater than 1 h). After reaction with trypsin, this alpha-macroglobulin cleared rapidly (t 1/2 = 3 min), and this clearance was inhibited by a 1000-fold molar excess of human alpha 2M-methylamine. Ovomacroglobulin-trypsin did not inhibit the binding of 0.2 nM 125I-labeled human alpha 2M-methylamine to mouse peritoneal macrophages in vitro, but chicken alpha M reacted with trypsin inhibited the binding by 50% at 1.9 nM. A kappa I of 1.1 nM was calculated for the binding of chicken alpha M-trypsin to the mammalian alpha-macroglobulin receptor. This affinity is comparable to that obtained with human and bovine alpha 2M.     

Uptake and degradation of 125I-labeled rat asialoorosomucoid by the perfused rat liver

The uptake and degradation of a homologous rat serum asialoglycoprotein, ¹²⁵I-asialoorosomucoid, and the effects on this metabolism by leupeptin, a proteinase inhibitor, were studied in the perfused rat liver. ¹²⁵I-Asialoorosomucoid was rapidly taken up by the liver ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 5.7}\text{min}$) and acid-soluble degradation products began to appear in the circulating perfusate medium after 20–30 min. These products accounted for 60–65% of the initially added radioactivity after 90 min of perfusion. The early events in the galactose-mediated uptake of ¹²⁵I-asialoorosomucoid were unchanged by the presence of leupeptin. However, the appearance of acid-soluble degradation products was greatly reduced when livers had been pretreated with the inhibitor (1.0 mg for 60 min). This effect corresponded with an increase in acid-precipitable material being located within the lysosomal-rich fraction from homogenates of leupeptin-treated livers. Leupeptin inhibited degradation of ¹²⁵I-asialoorosomucoid by approx. 85% relative to control values over 90 min of perfusion. Inhibition of asialoorosomucoid degradation was also demonstrated in vitro. Leupeptin (1.0 mM) reduced hydrolysis of this glycoprotein substrate by greater than 50% during a 24 h incubation with isolated lysosomal enzymes. The thiol proteinases, cathespin B, H and L, which are known to be inhibited by leupeptin, are apparently involved in initiating digestion of rat ¹²⁵I-asialoorosomucoid within liver lysosomes. As a result of inhibition by leupeptin both in the perfused liver and in vitro very limited changes occured in the native molecular weight of the starting glycoprotein.     

Diacytosis of 125I-asialoorosomucoid by rat hepatocytes. A non-lysosomal pathway insensitive to inhibition by inhibitors of ligand degradation

Diacytosis of 125I-asialoorosomucoid by rat hepatocytes was studied by preincubating the cells with the labelled ligand at 37 degrees C for 30 min or 18 degrees C for 2 h, washing free of cell surface receptor-bound tracer at 4 degrees C and then reincubating at 37 degrees C. The cells preloaded at 37 degrees C released a maximum of 18% of the total intracellular ligand as undegraded molecules after 1 h of incubation with an apparent first-order rate constant of 0.018 min-1 (t1/2 = 39 min). When the preloaded cells were incubated in the presence of 100 micrograms/ml unlabelled asialoorosomucoid or 5 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, the amount of the released ligand increased to 32 and 37%, respectively, without apparent change in kinetics, indicating that these agents prevented rebinding of the released ligand. In the presence of 5 microM colchicine, 20 microM cytochalasin B, 20 microM chloroquine, 10 mM NH4Cl, 10 microM monensin or 20 microM leupeptin, degradation of the preloaded ligand was inhibited, whereas the release of the ligand was either slightly increased or unchanged. Similar effects of leupeptin, colchicine and asialoorosomucoid were observed with cells preloaded at 18 degrees C. These results indicate that diacytosis of 125I-asialoorosomucoid occurs from a prelysosomal compartment via a route insensitive to inhibition by the inhibitors of ligand degradation.     

Purification and various properties of asialoglycoprotein receptors from the mouse liver

The receptor for asialoglycoproteins was isolated from murine liver and was purified by means of biospecific chromatography on sepharose-Asialo-orosomucoid. The obtained receptor with an absorption maximum at 277 nm binds to the nonreducing terminal galactosyl residues of glycoproteins similar to the receptors from liver of other mammalians. The interaction between this receptor and desialylated glycoproteins requires the presence of calcium. The dependence of specific binding on the concentration of [125I]acialo-orosomucoid used as a ligand gives a saturating curve. The dissociation constant for the receptor-ligand complex is 0.4 X 10(-9) M. Similar to asialo-orosomucoid, the receptor binds the p-aminophenyl-beta-D-galactopyranoside derivatives of bovine serum albumin, ovalbumin and acid alpha-glucosidase synthesized by us earlier. Possible use of the asialoglycoprotein receptor as a highly specific carrier transporting the modified acid alpha-glucosidase to hepatocyte lysosomes is discussed.     

Purification of vasoactive intestinal peptide receptor from porcine liver by a newly designed one-step affinity chromatography

Vasoactive intestinal peptide (VIP) receptors were solubilized from porcine liver membrane using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. The solubilized VIP receptor has been purified approximately 50,000-fold to apparent homogeneity by a one-step affinity chromatography using a newly designed VIP-polyacrylamide resin. The purified receptor bound 125I-VIP with a Kd of 22.3 +/- 0.7 nM and retained its peptide specificity toward VIP-related peptides. The specific activity of the purified receptor (16,400 pmol/mg of protein) was very close to the theoretical value (18,900 pmol/mg of protein) calculated assuming one binding site/protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified receptor revealed a single band with an Mr of 53,000 after either silver staining or radioiodination. Affinity labeling of the purified receptor with 125I-VIP using dithiobis(succinimidyl propionate) gave a single radioactive band, the labeling of which was completely inhibited by an excess of unlabeled VIP. In conclusion, an Mr 53,000 protein containing the VIP-binding site was purified to homogeneity by a one-step affinity chromatography using immobilized VIP.     

The rat liver insulin receptor

Using insulin affinity chromatography, we have isolated highly purified insulin receptor from rat liver. When evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, the rat liver receptor contained the Mr 125,000 alpha-subunit, the Mr 90,000 beta-subunit, and varying proportions of the Mr 45,000 beta'-subunit. The specific insulin binding of the purified receptor was 25-30 micrograms of 125I-insulin/mg of protein, and the receptor underwent insulin-dependent autophosphorylation. Rat liver and human placental receptors differ from each other in several functional aspects: (1) the adsorption-desorption behavior from four insulin affinity columns indicated that the rat liver receptor binds less firmly to immobilized ligands; (2) the 125I-insulin binding affinity of the rat liver receptor is lower than that of the placental receptor; (3) partial reduction of the rat liver receptor with dithiothreitol increases its insulin binding affinity whereas the binding affinity of the placental receptor is unchanged; (4) at optimal insulin concentration, rat liver receptor autophosphorylation is stimulated 25-50-fold whereas the placental receptor is stimulated only 4-6-fold. Conversion of the beta-subunit to beta' by proteolysis is a major problem that occurs during exposure of the receptor to the pH 5.0 buffer used to elute the insulin affinity column. The rat receptor is particularly subject to destruction. Frequently, we have obtained receptor preparations that did not contain intact beta-subunit. These preparations failed to undergo autophosphorylation, but their insulin binding capacity and binding isotherms were identical with those of receptor containing beta-subunit. Proteolytic destruction and the accompanying loss of insulin-dependent autophosphorylation can be substantially reduced by proteolysis inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

Purification and protein sequence analysis of rat liver prolactin receptor

Prolactin receptors were purified from rat liver membranes by single-step immunoaffinity chromatography using a specific monoclonal antibody to the rat liver prolactin receptor. Scatchard analysis of 125I-human growth hormone binding to the purified receptor revealed two classes of specific binding sites with Ka = 18.5 x 10(9) and 1.2 x 10(9) M-1. Considering that both classes of binding sites are responsible for high affinity prolactin binding, the partially purified receptor preparation had a binding activity of 1.69 nmol/mg protein, representing 1000-fold purification over microsomal receptors with a recovery of 52%. From three separate purifications, 6 mg of partially purified prolactin receptor were obtained with a purity of approximately 4 to 6.5%. Thus, the use of monoclonal antibody for affinity chromatography resulted in a large improvement of prolactin receptor purification compared to previous hormone affinity chromatography (300-fold purification, 15% recovery). The purified receptor was run on preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis, and a homogeneous preparation of prolactin receptor was obtained by electroelution from gel slices corresponding to Mr 38,000-43,000. Immunoblot analysis using a radiolabeled monoclonal antibody revealed two separate but closely located bands of Mr 42,000 and 40,000 in microsomal, partially purified, and electroeluted preparations. The homogeneous receptor protein was extensively digested with L-1-tosylamido-2-phenylethyl chloromethyl ketone trypsin, and 10 internal amino acid sequences of the rat liver prolactin receptor were determined by gas-phase sequence analysis. Oligonucleotide probes were prepared against two of these internal sequences, and a prolactin receptor cDNA was isolated from a rat liver library using one of these probes (Boutin, J. M., Jolicoeur, C., Okamura, H., Gagnon, J., Edery, M., Shirota, M., Banville, D., Dusanter-Fourt, I., Djiane, J., and Kelly, P. A. (1988) Cell 53, 69-77). The amino acid sequence deduced from the cDNA reveals three potential sites of N-linked glycosylation, two of which were confirmed during protein sequencing. The prolactin receptor was characterized by affinity labeling with 125I-human growth hormone. Cross-linking of microsomes revealed a single band for the hormone-receptor complex with Mr 62,000. On the other hand, cross-linking of Triton X-100-solubilized or partially purified receptor with labeled hormone resulted in the appearance of two bands with Mr 62,000 and 102,000, suggesting the existence of a subunit structure of the prolactin receptor, or alternatively, the existence of two types of prolactin receptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

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

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

Hepatic uptake of asialoglycoprotein is different among mammalian species due to different receptor distribution

Isolated hepatocytes of rat, rabbit and guinea pig were found to take up and degrade 125I-labelled asialoorosomucoid at different rates with the rank order: rabbit greater than rat greater than guinea pig. Measurement of 125I-asialoorosomucoid binding at 4 degrees C to these hepatocytes revealed that all these cells had two classes of receptors with a major difference occurring in the number of high-affinity binding sites. The average binding affinity constants (K) and receptor concentration (N) calculated from a least-square analysis of the Scatchard plots were K1 = 1.15.10(9) M-1, K2 = 0.93.10(7) M-1, N1 = 0.049 pmol/mg cell protein and N2 = 0.27 pmol/mg cell protein for the rat; K2 = 3.16.10(7) M-1, N1 = 0.027 pmol/mg cell protein and N2 = 0.13 pmol/mg cell protein for the guinea pig and K1 = 0.74.10(9) M-1, K2 = 3.85.10(7) M-1, N1 = 0.205 pmol/mg cell protein and N2 = 0.37 pmol/mg cell protein for the rabbit hepatocytes, respectively. Measurement of the total number of cellular receptors after solubilization with Triton X-100 also revealed the same receptor concentration rank order of rabbit (5.8 pmol/mg cell protein) greater than rat (0.55 pmol/mg cell protein) greater than guinea pig (0.18 pmol/mg cell protein). Intravenous injection of 125I-asialoorosomucoid into anesthetized animals of matched body weight also indicated that the rate of plasma clearance and the rate of appearance of the degraded product of the tracer were different among these species with the same rank order as that observed with isolated hepatocytes. Thus there is a fundamental difference in the number of asialoglycoprotein receptors both on the cell surface and inside hepatocytes of these mammalian species.     

Recycling of the asialoglycoprotein receptor and the effect of lysosomotropic amines in hepatoma cells

Receptor-mediated uptake and degradation of 125I-asialoorosomucoid (ASOR) in human hepatoma HepG2 cells is inhibited by the lysosomotropic amines chloroquine and primaquine. In the absence of added ligand at 37 degrees C, these amines induce a rapid (t1/2 5.5-6 min) and reversible loss of cell surface 125I-ASOR binding sites as well as a rapid decrease in 125I-ASOR uptake and degradation. There is no effect of these amines on the binding of 125I-ASOR to the cell surface at 4 degrees C or on the rate of internalization of prebound 125I-ASOR. The loss of 125I-ASOR surface binding at 37 degrees C is not attributable to altered affinity of ligand-receptor binding. In the presence of added ligand at 37 degrees C, there is a more rapid (t1/2 2.5-3 min) loss of hepatoma cell surface receptors. In addition, the amines inhibit the rapid return of the internalized receptor to the cell surface. We examined the nature of this loss of 125I-ASOR surface binding sites by following the fate of receptor molecules after biosynthetic labeling and after cell surface iodination. At 37 degrees C, chloroquine and primaquine induce a loss of asialoglycoprotein receptor molecules from the hepatoma cell surface to an internal pool.     

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

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

Studies with anti-growth hormone receptor antibodies.

Antisera against a partially purified growth hormone receptor derived from rabbit liver were generated in guinea pigs. The antisera specifically inhibited the binding of 125I-ovine growth hormone (oGH) to liver membranes but had no effect on the binding of 125I-ovine prolactin to rabbit mammary gland receptors. These antisera did not bind or destroy 125I-oGH. Moreover, the binding of labeled growth hormone to membrane particles derived from liver of several species was also inhibited by the antisera, thus suggesting that immunological determinants of the growth hormone receptor of several species are similar. gamma-Globulin fractions derived from the antisera were responsible for the inhibition. In addition 125I-gamma-globulin derived from one antiserum bound to membrane pellets with a corresponding decline in 125I-oGH binding. Kinetic analysis of inhibition of 125I-oGH binding suggested a hyperbolic competitive inhibition, a point of view which is favored by the demonstration of a hormone receptor . antibody complex. The availability of the antireceptor sera confirmed previous data that differential affinity chromatography separated growth hormone and prolactin receptors in solubilized rabbit liver membrane preparations. The antireceptor sera will be useful probes in further characterization of the growth hormone receptor.     

Purification and properties of human chorionic gonadotropin/lutropin receptor from plasma-membrane and soluble fractions of bovine corpora lutea.

Plasma-membrane and soluble fractions containing human chorionic gonadotropin/lutropin receptor were prepared from bovine corpora lutea by ultracentrifugation. The plasma-membrane and soluble fractions were studied for physicochemical properties, salts and gangliosides. The receptor preparations obtained from the plasma-membrane purified individually by sucrose-density-gradient centrifugation, which resulted in a partial dissociation of the hormone-binding subunit from the intact functional receptor unit, which consists of both hormone-binding (regulatory) and adenylate cyclase-associated (catalytic) subunits. The fractions containing the functional receptor unit were further purified by gel filtration on Sepharose-6B and chromatography on concanavalin A-Sepharose. The 'receptor' was finally purified by affinity chromatography on a column of controlled-pore glass covalently coupled to hu man chorionic gonadotropin. The purified receptor from the plasma-membrane and the soluble fractions contained binding capacities of 901000 and 87000 fmol of human chorionic gonadotropin/mg of protein. Yields of 0.02 and 0.22mg of protein were obtained from 250 g of bovine corpora lutea, which represents a 10000- and 1000-fold increase respectively in the specific binding with 125I-labelled human chorionic gonadotropin. Immunization of rabbits with a partially purified receptor fraction generated antibodies that specifically inhibited the binding of the 125I-labelled human chorionic gonadotropin to the receptor.     

Fibronectin-binding properties of the purified platelet glycoprotein IIb-IIIa complex

Fibronectin binds to specific receptors on the surface of washed, thrombin-activated platelets. Evidence suggests that these receptors are closely associated with the platelet glycoprotein IIb-IIIa complex (GP IIb-IIIa). To determine whether GP IIb-IIIa itself can form a platelet receptor for fibronectin, we used a filtration assay to examine the interaction of purified fibronectin with purified GP IIb-IIIa incorporated into phospholipid vesicles. 125I-Fibronectin binding to the phospholipid vesicles required the presence of incorporated GP IIb-IIIa and was specific, time-dependent, reversible, saturable, and divalent cation-dependent (Mg2+ greater than Ca2+). The dissociation constant for 125I-fibronectin binding to the GP IIb-IIIa-containing vesicles in the presence of 2 mM MgCl2 was 87 nM. Proteins or peptides that inhibit 125I-fibronectin binding to whole platelets also inhibited 125I-fibronectin binding to the GP IIb-IIIa vesicles. Thus, specific 125I-fibronectin binding was inhibited by excess unlabeled fibrinogen or fibronectin, the anti-GP IIb-IIIa monoclonal antibody 10E5, the decapeptide from the carboxyl terminus of the fibrinogen gamma-chain, and the tetrapeptide Arg-Gly-Asp-Ser from the cell-binding domain of fibronectin. In contrast to results obtained using whole platelets, unlabeled fibronectin inhibited 125I-fibronectin binding to the GP IIb-IIIa vesicles. These results show that 125I-fibronectin binds directly to purified GP IIb-IIIa with most of the previously reported properties of 125I-fibronectin binding to washed, thrombin-stimulated platelets. Thus, GP IIb-IIIa has the potential to function as a platelet receptor for fibronectin as well as for fibrinogen.     

Multiplicity of receptors for vasoactive intestinal polypeptide (VIP): differential effects of apamin on binding in brain, uterus and liver

Apamin is a neurotoxic octadecapeptide from bee venom, which has been shown to inhibit the non-adrenergic, non-cholinergic inhibitory innervation of the smooth muscle of the gut. Since vasoactive intestinal polypeptide (VIP) has been proposed as a possible inhibitory neurotransmitter, the effect of apamin on the receptor binding of 125I-VIP was studied using the following assays: (1) isolated synaptosomes from rat cerebral cortex, (2) crude plasma membranes from hog uterine smooth muscle, and (3) purified plasma membranes and isolated hepatocytes from hog liver. Apamin inhibited the receptor-bound 125I-VIP on membranes from brain or myometrium, although the binding affinity was 100-1000 times lower than for VIP. The displacement curves for VIP and apamin were parallel suggesting that apamin interacts with both the low and high affinity VIP receptors. In membranes and cells from liver, apamin was unable to displace receptor-bound 125I-VIP in concentrations up to 50 mumol/l. The findings suggest that the VIP receptors in liver are different from those in the brain cortex and myometrium.