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

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

Purification of the type II insulin-like growth factor receptor from rat placenta

The membrane receptor for insulin-like growth factor II (IGF II) has been purified to near homogeneity from rat placenta by chromatography of crude plasma membranes solubilized in Triton X-100 on agarose-immobilized IGF II. Elution of the IGF II receptor from the matrix at pH 5.0 in the presence of 1.5 M NaCl resulted in a receptor purification of 1100-fold from isolated plasma membranes, or 340-fold from the Triton extract with an average yield of about 50% in five separate purifications. Analysis of 125I-IGF II binding to the solubilized receptor in the Triton extract and in purified form by the method of Scatchard demonstrated no change in receptor affinity (Kd = 0.72 nM). Sodium dodecyl sulfate electrophoresis of the purified receptor showed one major band at Mr = 250,000 with only minor contamination. Affinity labeling of the receptor in isolated placenta membranes and in purified form using 125I-IGF II and the cross-linking agent disuccinimidyl suberate resulted in covalent labeling of only the Mr = 250,000 band. Such labeling was abolished by unlabeled IGF II but was unaffected by insulin, consistent with the previously reported specificity of IGF II receptor (Massague, J., and Czech, M.P. (1982) J. Biol. Chem. 257, 5038-5045). These results establish a one step affinity method for the purification of the type II IGF receptor that is rapid and highly efficient.     

Purification of the insulin-like growth factor II (IGF-II) receptor from an IGF-II-producing cell line, and generation of an antibody which both immunoprecipitates and blocks the type 2 IGF receptor

18,54-SF cells, which secrete rat insulin-like growth factor II (rIGF-II), have abundant type 2 IGF receptors. We have purified the type 2 receptor from these cells by solubilization of crude membranes in Triton X-100, followed by chromatography on agarose-immobilized rIGF-II. A partially purified receptor preparation, obtained by chromatography of solubilized membranes over wheat germ agglutinin, was used to immunize a rabbit. The antibody generated both immunoprecipitates the type 2 receptor, and specifically inhibits IGF-II binding to a variety of rat tissues, including 18,54-SF cells, BRL-3A cells and placenta. The presence of abundant type 2 receptors on an rIGF-II-secreting cell line is consistent with an autocrine role for IGF-II in select cells.     

Comparison of solubilized and purified plasma membrane and nuclear insulin receptors

Prior studies have detected biochemical and immunological differences between insulin receptors in plasma membranes and isolated nuclei. To further investigate these receptors, they were solubilized in Triton X-100 and partially purified by wheat germ agglutinin-agarose chromatography. In these preparations, the nuclear and plasma membrane receptors had very similar pH optima (pH 8.0) and reactivities to a group of polyclonal antireceptor antibodies. Further, both membrane preparations had identical binding activities when labeled insulin was competed for by unlabeled insulin (50% inhibition at 800 pM). Next, nuclear and plasma membranes were solubilized and purified to homogeneity by wheat germ agglutinin-agarose and insulin-agarose chromatography. In both receptors, labeled insulin was covalently cross-linked to a protein of 130 kilodaltons representing the insulin receptor alpha subunit. When preparations of both receptors were incubated with insulin and then adenosine 5'-[gamma-32P]triphosphate, a protein of 95 kilodaltons representing the insulin receptor beta subunit was phosphorylated in a dose-dependent manner. These studies indicate, therefore, that solubilized plasma membrane and nuclear insulin receptors have similar structures and biochemical properties, and they suggest that they are the same (or very similar) proteins.     

The mechanisms of up-regulation of the hepatic insulin receptor in hypoinsulinemic diabetes mellitus

The mechanism underlying the increased insulin binding found in hepatic plasma membranes from streptozotocin-diabetic rats was evaluated by measuring insulin binding to intact and Triton X-100-soluble extracts of plasma membranes prepared from the livers of control rats and rats administered streptozotocin (85 mg/kg). In addition, to assess whether the cellular content of hepatic insulin receptors is also increased in diabetic animals, we measured insulin-binding activity in intact and soluble extracts of total hepatic cellular membrane preparations (100,000 X g cellular pellets). The data indicate that while insulin binding is increased (52 +/- 3%) in intact hepatic plasma membranes from diabetic rats compared to control rats, there is no comparable increase in insulin binding in intact total cellular membranes or in Triton X-100-soluble extracts of plasma membranes or total cellular membranes. We therefore conclude that the enhanced insulin binding found in the livers of diabetic rats is the result of a local redistribution of plasma membrane insulin receptors from cryptic to exposed sites. Finally, the data suggest the presence of a negative modulator of insulin-binding affinity in intact plasma and total cellular membranes.     

Homogeneous functional insulin receptor from 3T3-L1 adipocytes. Purification using N alpha B1-(biotinyl-epsilon-aminocaproyl)insulin and avidin-sepharose

Insulin receptor was purified 10,000-fold from cultured mouse 3T3-L1 adipocytes in 35% overall yield. The specific activities of 125I-insulin binding and autophosphorylation increased in parallel, following the initial Triton X-100 extraction of membranes. The isolation protocol, performed entirely at pH 8.45, entailed adsorption by avidin-Sepharose CL-4B of a complex formed between Triton X-100-solubilized insulin receptor and N alpha B1-(biotinyl-epsilon-aminocaproyl)insulin, and the specific elution of the complex with biotin. The avidin-Sepharose CL-4B was a partially denatured preparation, showing estimated dissociation constants of 0.2 microM for biotin and approximately 1 microM for the bifunctional ligand at, pH 7, 4 degrees C. The bifunctional ligand was characterized by 70% competency in binding to avidin, 100% competency in binding to solubilized insulin receptor, full stimulation of autophosphorylation of the isolated receptor, and maximal stimulation of hexose uptake by intact 3T3-L1 adipocytes. The insulin binding properties of the insulin receptor were uniform throughout this purification procedure. At pH 8.45, 4 degrees C, an average Kd = 0.72 nM was determined for a single class of noninteracting insulin binding sites. The apparent autophosphorylation of the beta-subunit was also unchanged following affinity chromatography. A single oligomeric structure was established for the purified receptor, composed only of 135,000- and 95,000-Da subunits, whose association was lost by denaturation in the presence of reducing agent. This single structure occurred in the initial Triton X-100 extract. The purified insulin receptor was capable of autophosphorylating the beta-subunit and catalyzed phosphorylation of protein substrates.     

Properties and partial purification of the detergent-solubilized insulin receptor A demonstration of negative cooperativity in micellar solution

Turkey erythrocytes possess insulin receptors with binding properties very similar to those of mammalian insulin receptors. In the present study, the insulin receptor of the avian erythrocyte has been solubilized in Triton X-100, extensively characterized and partially purified, and its properties compared to those of the membrane-bound receptor.The solubilized insulin receptor has a Stokes radius of 70 Å and an apparent molecular weight of 300 000 in 0.05% Triton. The binding of insulin to the soluble receptor was very similar to the binding observed with the membrane-bound receptor. Thus, binding was markedly temperature dependent for both the soluble and membrane-bound forms, although the kinetics of binding were slower with the soluble receptor. Both forms of the receptor also showed a sharp pH optimum; however, solubilization produced a shift from maximal binding at pH 7.8 to pH 7.3. The soluble receptor also retained insulin analog specificity, ion sensitivity and negative cooperativity. The soluble receptor did not appear to degrade either bound or free insulin.On DEAE-cellulose chromatography the receptor eluted as a single peak. The specific activity of this partially purified preparation was 25–30 pmol/mg protein (about 500-fold enrichment over crude extract and 5-fold over highly purified membranes). Extensive attempts to purify further the receptor by gel filtration, carboxymethyl-cellulose chromatography and affinity chromatography resulted in either a very low yield or only modest enrichment. Purification was also complicated because the receptor was easily denatured; about 40% of the activity was lost after a 90-min exposure to 3 M urea or pH 4.5.     

Insulin binding and insulin receptor tyrosine kinase activity are not altered in the liver of rats with non-insulin-dependent diabetes

Using the insulin-glucose clamp technique, we have previously shown that an increased sensitivity to insulin in vivo is a characteristic of the liver in rats with non-insulin-dependent diabetes induced by neonatal streptozotocin administration. We have thus studied the properties of liver insulin receptor in that model. 125I-porcine insulin binding was found normal both in isolated plasma membranes and in solubilized, wheat germ agglutinin purified receptors prepared from livers of rats with non-insulin-dependent diabetes, when compared to controls. Basal and insulin-stimulated insulin receptor kinase activities were also found normal for both the autophosphorylation of the beta subunit of the insulin receptor and the phosphorylation of the artificial substrate poly (Glu-Tyr) 4:1. Thus, in that model of chronic insulin deficiency and mild hyperglycemia: 1) liver insulin receptors are not up-regulated; 2) tyrosine kinase activity remains unaffected. This last observation supports the hypothesis that the increased insulin effect in the liver of rats with non-insulin-dependent diabetes is probably distal to the insulin receptor kinase.     

Detergents affect insulin binding, tyrosine kinase activity and oligomeric structure of partially purified insulin receptors.

Insulin receptor activities, i.e., insulin binding and tyrosine kinase activation depend on the lipid environment of the receptor. As detergent may disrupt or interfere with this environment, we investigated the effect of various common detergents on insulin receptor properties. Experiments were carried out (i) on solubilized and partially purified insulin receptor and (ii) on the receptor reconstituted into phosphatidylcholine vesicles. The detergents tested, Triton X-100, octyl-beta-D-glucopyranoside, octyl-beta-D-thioglucopyranoside, 3[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid (Chaps), and Na deoxycholate affected the insulin receptor properties differently when compared with the control receptor in the absence of detergent. On the partially purified insulin receptor, Na deoxycholate inhibited both insulin receptor activities; octyl-beta-D-glucopyranoside and octyl-beta-D-thioglucopyranoside decreased insulin binding and kinase activation as their concentration increased, particularly above their respective critical micellar concentration (CMC). Triton X-100 was the only detergent which allowed an increase of insulin binding and kinase activation throughout the whole range of concentrations assayed. Reconstitution of the receptor into phosphatidylcholine vesicles protected the receptor from the direct effects of the detergents, for both the stimulation observed with Triton X-100 and the inhibition produced by the other detergents. In order to determine the effect of detergents on the oligomeric forms of the soluble insulin receptor, we investigated a new rapid sucrose gradient centrifugation technique. Insulin receptors were detected on the gradient by 125I insulin binding. For low concentrations of detergent, i.e., near the CMC, octylglucoside, Chaps, and Triton X-100 favored the (alpha 2 beta 2)2 oligomeric form of the receptor. Higher concentrations of Triton X-100 did not modify the polymeric state of the receptor. In contrast, octylglucoside and Chaps induced an increase in the sedimentation coefficient of the receptor which appeared as (alpha 2 beta 2)3 and (alpha 2 beta 2)4 forms. These alterations in the oligomerization status of the insulin receptor may explain the deleterious effects observed with both Chaps and octylglucoside at higher concentrations.     

The human placental transferrin receptor: Reconstitution into liposomes and electron microscopy

Human transferrin receptor was isolated from Triton X-100 solubilized placental plasma membranes by a rapid one-step chromatographic procedure based on immunoadsorption of the receptortransferrin complex on anti-transferrin Sepharose and lectin-affinity on wheat germ agglutinin. Following exchange of Triton X-100 with CHAPS or n-octylglucoside, the purified receptor was incorporated into egg phosphatidylcholine liposomes upon, detergent removal by dialysis (lipid/protein ratio 15:1 to 45:1 (w/w) Reconstitution of the receptor was confirmed by trypsin cleavage to dissociate the large extracellular receptor domain from the liposomal membranes. Electron micrographs of the receptor-lipid recombinants negatively stained with sodium sillicotungstate, showed ographs of the receptor-lipid recombinants negatively stained with sodium sillicotungstate, showed that the receptor molecules distributed very inhomogeneously on the liposomes, most receptors being clustered. Single copies of the receptor were seen as elongate structures (5×10 nm) oriented with their long axis parallel to the liposome surface and separated from this by a 2–3 nm gap. This result provides evidence for a narrow connecting link between the globular extracellular receptor domain and the membrane spanning segment.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate - PAGE polyacrylaminde gel electrophoresis - PC phosphatidylcholine - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate - WGA wheat germ agglutinin     

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.     

The porcine LH/hCG receptor. Characterization and purification

Porcine luteal LH/hCG receptor (LH/hCG R) was solubilized with 70-80% recovery from the crude plasma membrane fraction by Triton X-100 in the presence of 25% glycerol and protease inhibitors. The solubilized receptor maintained 90% of original activity at -60 degrees C for 90 days. Equilibrium association constant (Ka) values of 1.92, 2.22, and 2.03 X 10(10) M-1 were observed for the whole homogenate, plasma membrane fraction, and solubilized LH/hCG R preparations, respectively. The specific binding capacity for the same fractions were 49, 70, 55 fmol/mg protein, respectively. Complexes of LH/hCG R and Triton X-100 were resolved into two components with approximate Mr = 2.7 X 10(5) and 5.4 X 10(5) by gel filtration on Sepharose 6B and two glycoprotein components by chromatography on concanavalin A-Sepharose. Solubilized porcine LH/hCG R was purified by two cycles of affinity chromatography on highly purified hCG-Sepharose with an overall recovery of 30-35% of the initial activity in the Triton extract. Purified porcine LH/hCG R had a specific binding capacity of 2300 pmol/mg protein and a Ka = 1.5 X 10(10) M-1. Silver staining of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels demonstrated that the major protein in porcine LH/hCG R preparations has Mr = 68,000. A weakly staining band at Mr = 45,000 was also observed in the purified receptor preparation. Analysis of iodinated purified LH/hCG R by autoradiography has confirmed these results. Porcine LH/hCG R was purified 40,000-fold by this method.     

Chronic Intracerebroventricular Infusion of Insulin Failed to Alter Brain Insulin-Binding Sites, Food Intake, and Body Weight

The present study was performed to explore the role of exogenous insulin in CSF in the control of energy balance in the rat. For this purpose, adult male Sprague-Dawley rats carrying an indwelling cannula in the right lateral cerebral ventricle were infused for a maximum of 10 days with insulin (Actrapid) at various rates (starting at 0, 45, 85, 170, and 600 ng/day) or anti-insulin antibody (IgG fraction; diluted 1:10 wt/vol) with an osmotic minipump. All those treatments did not modify the growing rates; neither total daily food intake nor the circadian rhythm of food intake was further modified. The chronic insulin infusion starting at 600 ng/day resulted in a chronic significant increase in CSF insulin levels without changing the plasma insulin level. It failed to alter specific insulin binding sites to Triton X-100 solubilized microsomal membranes from various brain areas (cerebral cortex, olfactory bulbs, and lateral and medial hypothalami) at the end of the 5- or 10-day period of insulin infusion. Purification of insulin receptors on a wheat germ agglutinin did not reveal any further effect of insulin. From these results, it seems unlikely that the input to the brain insulin-effector systems could arise from CSF insulin.     

Purification and properties of a membrane-bound insulin binding protein, a putative receptor, from Neurospora crassa.

The protein that is responsible for specific, high-affinity binding of insulin to the surface of Neurospora crassa cells has been purified to homogeneity. The insulin binding activity of solubilized plasma membranes resembled that of intact cells with regard to affinity of binding, specificity for mammalian insulins, and amount of insulin bound per cell. Insulin binding activity was purified from Triton X-100 solubilized membranes in two steps: FPLC on a MonoQ HR5/5 column; and affinity chromatography on insulin-agarose. The pure material migrated as a single band of ca. 66 kDa on SDS gels, pI = 7.4 by isoelectric focusing. The protein bound 5.34 pmol of insulin/micrograms, or 35% of that expected for univalent binding. Cross-linking of 125I-insulin to pure protein or to solubilized membranes revealed a single labeled band of 67-70 kDa on SDS gels. In nonreducing native gels, two labeled bands of ca. 55 and 110 kDa were produced after cross-linking, and two bands of similar molecular weight bound iodinated insulin after transfer to nitrocellulose filters. These may correspond to active monomer and dimer forms. The pure protein possessed no protein kinase activity against itself, or against exogenous substrates (histone H2, casein, or the synthetic peptide Glu80-Tyr20), and possessed no detectable phosphorylated amino acids. It is suggested, however, that this 66-kDa protein is the "receptor" that mediates insulin-induced downstream metabolic effects.     

Characterization of a membrane protein from brain mediating the inhibition of inositol 1,4,5-trisphosphate receptor binding by calcium.

Inositol 1,4,5-trisphosphate (InsP3) is a component of the phosphoinositide second-messenger system which mobilizes Ca2+ from intracellular stores. Recently, an InsP3 receptor binding protein from rat cerebellar membranes was solubilized and purified to homogeneity. The potent inhibition by Ca2+ of [3H]InsP3 binding to the InsP3 receptor in cellular membranes is not apparent in the purified receptor. The Ca2+-dependent inhibition of [3H]InsP3 binding in the crude homogenate (concn. giving 50% inhibition = 300 nM) can be restored by addition of solubilized cerebellar membranes to the purified receptor. In the present study, we further characterize the protein in solubilized membranes which confers Ca2+-sensitivity to the receptor, and which we term 'calmedin'. Calmedin appears to be a neutral membrane protein with an estimated Mr of 300,000 by gel filtration in the presence of Triton X-100. Calmedin confers a Ca2+-sensitivity to InsP3 receptor binding, which can be completely reversed by 10 min incubation with EDTA and therefore does not represent Ca2+-dependent proteinase action. Calmedin effects on the purified InsP3 receptor depend on Ca2+ binding to the calmedin, although Ca2+ also binds directly to the InsP3 receptor. The regional distribution of calmedin differs from that of the InsP3 receptor in the brain, suggesting that it also mediates other Ca2+-dependent functions. Calmedin activity in peripheral tissues is much lower than in brain.     

Reconstitution of pure acetylcholine receptor in phospholipid vesicles and comparison with receptor-rich membranes by the use of a potentiometric dye

Acetylcholine receptor, isolated in Triton X-100 on a cobra alpha-neurotoxin affinity column was incorporated into unilamellar phospholipid vesicles by a detergent depletion method using Amberlite XAD-2. Vesicles of an average diameter of 25 nm were formed, as verified by freeze-fracture electron microscopy and gel filtration. 85 to 95% of the alpha-bungarotoxin binding sites of the reconstituted acetylcholine receptor were oriented towards the outside of the vesicles. In the reconstituted receptor one molecule of residual Triton X-100 per 2.5 alpha-bungarotoxin binding sites on the receptor molecule could be assessed. The reconstituted protein was not accessible to papain digestion, whereas the pure acetylcholine receptor, solubilized by Triton X-100 was split into smaller polypeptides under the same condition. Reconstituted acetylcholine receptor and receptor-rich membranes did not exhibit the same behavior as measured by use of a potentiometric dye. This is interpreted as an irreversible alteration of at least 95% of the receptors purified in the presence of Triton X-100. Furthermore, it could be shown that the fluorescence intensity changes induced by carbamylcholine in receptor-rich membranes did not reflect ion fluxes, but conformational changes of the protein or a displacement of the dye from the protein.     

The glycocalicin portion of platelet glycoprotein Ib expresses both high and moderate affinity receptor sites for thrombin. A soluble radioreceptor assay for the interaction of thrombin with platelets

A soluble radioreceptor assay has been developed to characterize thrombin receptor activities of the human platelet membrane. 125I-Thrombin was added to platelet membranes solubilized in 1% Triton X-100, and thrombin bound to platelet receptors was separated from free thrombin by precipitation with wheat germ agglutinin (WGA) in the presence of alpha 1-acid glycoprotein as carrier. Both high affinity binding (Ki, 0.09 nM; R1, 0.30 pmol/mg protein) and moderate affinity binding (K2, 38 nM; R2, 72 pmol/mg protein) were detected in the detergent-solubilized membrane preparations and these binding parameters were in excellent agreement with values previously determined using intact platelets (Harmon, J. T., and Jamieson, G. A. (1985) Biochemistry 24, 58-64). Using the soluble radioreceptor assay, both high and moderate affinity binding was detected in highly purified preparations of glycoprotein Ib (GPIb) and glycocalicin, and the binding isotherms were identical with those of the crude detergent-solubilized membrane preparation. Treatment of detergent-solubilized membranes with increasing concentrations of a monospecific polyclonal antibody to glycocalicin resulted in the stepwise depletion of GPIb and concomitant reductions of thrombin binding activity. These results demonstrate that both high and moderate affinity binding of thrombin to platelets is completely expressed in the glycocalicin portion of GPIb.     

Functional and physical molecular size of the chicken hepatic lectin determined by radiation inactivation and sedimentation equilibrium analysis

Radiation inactivation and sedimentation equilibrium analysis were used to determine the functional and physical size of the chicken hepatic membrane receptor that binds N-acetylglucosamine-terminated glycoproteins. Purified plasma membranes from chicken liver were irradiated with high energy electrons and assayed for 125I-agalactoorosomucoid binding. Increasing the dose of ionizing radiation resulted in a monoexponential decay in binding activity due to a progressive loss of binding sites. The molecular mass of the chicken lectin, determined in situ by target analysis, was 69,000 +/- 9,000 Da. When the same irradiated membranes were solubilized in Brij 58 and assayed, the binding protein exhibited a target size of 62,000 +/- 4,000 Da; in Triton X-100, the functional size of the receptor was 85,000 +/- 10,000 Da. Sedimentation equilibrium measurements of the purified binding protein yielded a lower limit molecular weight of 79,000 +/- 7,000. However, the solubilized lectin was detected as a heterogeneous population of oligomers with molecular weights as high as 450,000. Addition of calcium or calcium plus N-acetylglucosamine decreased the higher molecular weight species, but the lower limit molecular weights remained invariant. Similar results were determined when the chicken lectin was solubilized in Brij 58, C12E9, or 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid (CHAPS). Results from the present study suggest that in the plasma membrane, the functional species of the chicken hepatic lectin exists as a trimer. However, in detergent solution, the purified receptor forms a heterogeneous population of irreversible oligomers that exhibit binding activity proportional to size.     

Characterization and solubilization of cyclo(His-Pro) binding from rat liver membranes

We have found cyclo(His-Pro) binding in rat liver plasma membranes. This study focused on the characterization of solubilized binding for cyclo(His-Pro) in rat liver membranes. The cyclo(His-Pro) binding of liver membranes was solubilized by digitonin and octyl-glucopyranoside. The efficiency of solubilization with digitonin was greater. However, cyclo(His-Pro) binding was not solubilized by Triton X-100, CHAPS, or Lubrol. Digitonin-solubilized membranes showed cyclo(His-Pro) binding with a high affinity constant (17 nM) and a low binding capacity (38 fmol/mg protein). Lectins from wheat germ, Bandeiraea simplicifolia II, Dolichos biflorus, Glycine max, and Tetragonolobus purpureas significantly adsorbed [3H]cyclo(His-Pro)-binding complex, but Bandeiraea simplicifolia I, Ricinus communis I, or Lens culinaris did not adsorb the binding complex. An analysis of [3H]cyclo(His-Pro)-associated membranes by high performance gel filtration chromatography showed a radioactive peak of Mr 200,000. These data indicate that cyclo(His-Pro) binding of rat liver membranes is solubilized by digitonin and is a glycoprotein of Mr 200,000.     

High affinity binding proteins for pancreatic polypeptide on rat liver membranes.

We report here the identification on rat liver plasma membranes and microsomes of proteins that bind pancreatic polypeptide (PP) with high affinity and specificity (plasma membranes: KD = 4.6 nM, Bmax = 3.28 pmol/mg protein; microsomes: KD = 3.45 nM, Bmax = 18.7 pmol/mg protein). These binding proteins appeared coupled to a G-protein, since 0.1 mM guanosine 5'-O-(3-thiotriphosphate) decreased the affinity by half. When 125I-labeled PP-binding protein complexes covalently cross-linked with disuccinimido suberate were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two radioactive bands with M(r) values of 52,000 and 38,000 were demonstrated. Both bands were inhibited by unlabeled PP with an IC50 of approximately 5 nM (but not by neuropeptide Y or peptide YY). After the cross-linked complexes were solubilized from liver microsomes with 0.2% Triton X-100 and gel-filtered, they did not interact with the lectins wheat germ agglutinin, Ulex europaeus agglutinin, Ricinus communis agglutinin, and soy bean agglutinin. That these binding proteins may not be glycosylated was further supported by the failure of either peptide N-glycosidase F and endo-beta-N-acetylglucosaminidase F to alter the size of the PP-binding protein complexes on gel electrophoresis. These PP-binding proteins may serve as receptors and mediate a hepatic effect of PP.