Purification and characterization of neurotensin receptor from rat brain with special reference to comparison between newborn and adult age rats.

Scatchard analysis of saturation curves was performed to compared newborn and adult rat neurotensin receptor using [3H] neurotensin as a tracer. The membrane fraction of newborn rat cerebral cortex has a single population of neurotensin receptor (Kd = 0.13 nM, Bmax = 710 fmol/mg protein), whereas adults have two distinct neurotensin binding sites (high affinity site, Kd1 = 0.13 nM; low affinity site, Kd2 = 20 nM). High affinity neurotensin receptor, solubilized with digitonin, was purified from newborn rat cortex by affinity chromatography. An overall purification of 14,000-fold was achieved. The binding of [3H] neurotensin to the purified receptor is saturable and specific, with a Kd of 0.45 nM. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of 2-mercaptoethanol revealed purified material of a single major band of Mr = 55,000.     

High affinity angiotensin II receptors in myocardial sarcolemmal membranes. Characterization of receptors and covalent linkage of 125I-angiotensin II to a membrane component of 116,000 daltons

High affinity receptors for angiotensin II have been identified on purified cardiac sarcolemmal membranes. Equilibrium binding studies were performed with 125I-labeled angiotensin II and purified sarcolemmal vesicles from calf ventricle. The curvilinear Scatchard plots were evaluated by nonlinear regression analysis using a two-site model which identified a high affinity site Kd1 = 1.08 +/- 0.3 nM and N1 = 52 +/- 10 fmol/mg of protein and a low affinity site Kd2 = 52 +/- 16 nM and N2 = 988 +/- 170 fmol/mg of protein. Monovalent and divalent cations inhibited the binding of 125I-angiotensin II by 50%. The affinity of angiotensin II analogs for the receptor was determined using competitive binding assays; sarcosine, leucine-angiotensin II (Sar,Leu-angiotensin II), Kd = 0.53 nM; angiotensin II, Kd = 2.5 nM; des-aspartic acid-angiotensin II, Kd = 4.81 nM; angiotensin I, Kd = 77.6 nM. There is a positive correlation between potency in inducing positive inotropic response in myocardial preparations reported by others and potency for the hormone receptor observed in the binding assays. Pseudo-Hill plots of the binding data showed that agonists display biphasic binding with Hill numbers around 0.65 while antagonists recognized a single class of high affinity receptors with Hill numbers close to unity. These data were confirmed using 125I-Sar,Leu-angiotensin II in equilibrium binding studies which showed that this antagonist bound to a single class of receptor sites; Kd = 0.42 +/- 0.04 nM and N = 1050 +/- 110 fmol/mg of protein. Competition-binding experiments with this 125I-peptide yielded monophasic curves with Hill numbers close to unity for both agonists and antagonists. Membrane-bound 125I-angiotensin II was covalently linked to its receptor by the use of bifunctional cross-linking reagents such as dithiobis(succinimidyl propionate) and bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone. Analysis of the membranes showed the labeling of a component with an apparent Mr = 116,000. The affinity labeled species showed characteristics expected of a functional component of the high affinity receptor. The affinity labeling of this membrane component was inhibited by nanomolar angiotensin II or Sar,Leu-angiotensin II. Together these data indicate that high affinity receptors exist for angiotensin II that most likely mediate the positive inotropic effects of this hormone on myocardial cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pharmacology and molecular identification of vasoactive intestinal peptide (VIP) receptors in normal and cancerous gastric mucosa in man

In human antral membranes, VIP and its natural analogs inhibited the binding of HPLC-purified 125I-VIP, according to the following order of potency: VIP greater than rh GRF greater than helodermin greater than r PHI greater than PHM greater than p PHI greater than hp GRF greater than h, p secretin. No specific binding was detected in plasma membranes purified from the human fundus. In human antral membranes, Scatchard plots were compatible with the existence of two classes of VIP receptors, the first class with high affinity and low binding capacity (Kd = 0.1 nM, Bmax = 10 fmol/mg protein) and another class with a low affinity and higher binding capacity (Kd = 12) nM, Bmax = 1,000 fmol/mg protein). The structure of the VIP receptor in purified plasma membranes prepared from human antral glands and from the HGT-1 human gastric cancer cells was subsequently probed using the cross-linking reagent DSP and 125I-VIP. In agreement with the pharmacological study and the Scatchard analysis of the binding data, SDS gel electrophoresis of the solubilized receptor identified two radiolabeled peptides Mr 67,000 and 34,000 containing disulfide bonds. According to its sensitivity to low doses of VIP and to GTP, the Mr 67,000 binding site represents the membrane domains involved in the physiologial regulation of adenylate cyclase by VIP in normal and transformed human gastric epithelia.     

Two types of inositol trisphosphate binding in cardiac microsomes.

Two distinct types of [3H]IP3 binding were found in canine cardiac microsomes with high (Kd = 21 nM, Bmax = 0.66 pmol/mg) and low affinity (Kd = 230 nM, Bmax = 2.9 pmol/mg). Also found were low affinity [3H]IP4 binding (Kd = 190 nM, Bmax = 4.5 pmol/mg) and high affinity [3H]IP6 binding (Kd = 10 nM, Bmax = 4.9 pmol/mg). The rank order of potency to displace these radioligands indicates that binding of IP3 and IP6 is ligand-specific. Sucrose gradient centrifugation of the detergent-solubilized cardiac microsomes indicates that the molecular size of the cardiac high affinity IP3 receptor is similar to that of the aortic smooth muscle IP3 receptor and smaller than that of the ryanodine receptor which migrates more rapidly. The IP4 and IP6 binding migrates more slowly than the IP3 receptor.     

Purification and characterization of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells

The bombesin/gastrin-releasing peptide (GRP) receptor was solubilized from Swiss mouse 3T3 cell membranes in an active form and was purified about 90,000-fold to near homogeneity by a combination of wheat germ agglutinin-agarose and ligand affinity chromatography. The purified receptor displayed a single diffuse band with a Mr of 75,000-100,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After treatment of the receptor with N-glycanase, removing N-linked oligosaccharide moieties, the protein yielded a Mr = 38,000 band. These results agree with the Mr value estimated for the GRP receptor that was labeled on Swiss 3T3 cells by cross-linking to 125I-GRP1-27. GRP1-27 bound to the purified receptor with a Kd of 0.038 +/- 0.019 nM. By comparison, the soluble receptor in unfractionated extracts and intact membranes displayed a Kd for GRP1-27 of 0.036 +/- 0.003 nM and 0.13 +/- 0.04 nM, respectively. The relative potencies of a series of GRP analogs for the soluble receptor and intact membranes indicated that the extraction procedure did not significantly alter the receptor's ligand binding specificity. However coupling of the receptor to its guanyl nucleotide regulatory protein was not maintained in the soluble extract, and a G-protein did not co-purify with the receptor. Physiological concentrations of NaCl greatly inhibited the binding of some GRP analogs to the receptor, while the binding of other analogs was not affected. A domain on the GRP molecule involving Lys-13 or Arg-17 was identified which promoted binding to the GRP receptor under conditions of low ionic strength. These findings aided the development of an effective ligand affinity resin for the purification of the GRP receptor.     

Characterization of inositolpolyphosphate binding to myocardial membranes

Although it is well-accepted that the phosphatidylinositol signalling transduction pathway, producing inositol-1,4,5-P3 (InsP₃) and inositol-1,3,4,5-P₄ (InsP4) as second messengers, functions in heart muscle, virtually nothing is known about the roles of the higher inositol polyphosphates such as inositolhexakisphosphate (InsP₆). This study demonstrates that InSP₆ has the ability to bind intracellularly, with different binding characteristics, to different myocardial membranes. Binding to purified sarcoplasmic reticulum (SR) membranes, purified sarcolemmal (SL) membranes as well as to viable mitochondria were characterized. Binding to all these membranes display high as well as low affinity binding sites, with differing affinities. Kd values of binding to SR were 32 and 383 nM, to SL 61 and 1312 nM, while those of mitochondrial binding were 230 and 2200 nM respectively.InsP₄ binding was also investigated and displayed the following characteristics: to SR, one low affinity binding site (Kd = 203 nM) and to SL, a high as well as a low affinity binding site with Kd values of 41 and 2075 nM respectively. Presence of InsP₃, the second messenger for SR calcium release, at concentrations of 1 nM, elevated the binding of InsP₄ to SR and SL by a mean of 30% and 20% respectively.Fractionation of SR and SL membranes on sucrose density gradients, after solubilization with CHAPS, indicated that InsP₆ bound to two separate protein peaks in both these membranes, while InsP₄ bound to only one. In SR membranes, InsP₄ bound preferentially to a protein separating at high sucrose density while it bound to a protein separating at low sucrose density in SL membranes.     

Purification and characterization of the adenosine A2-like binding site from human placental membrane

We have purified and characterized the adenosine A2-like binding site from human placental membranes. 5'-N-Ethylcarboxamido[2,8-3H]adenosine ([3H]NECA) binds to this site, with a Kd of 240 nM and a Bmax of 13.0 pmol/mg in human placental membranes. The adenosine A2-like binding site was purified after extraction from placental membranes with 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. The purification included ammonium sulfate precipitation and concanavalin A, DEAE-Sephadex, and Sepharose 6B gel filtration chromatographies. The protein was purified 127-fold to homogeneity, with a final specific activity of 1.5-1.9 nmol/mg of protein and a 5.5-8.1% yield of binding activity from the membranes. The purified protein had similar binding properties and an identical potency order for displacement of [3H] NECA by adenosine analogs as the initial membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified protein revealed a single band at 98 kDa which coeluted with [3H]NECA binding activity during Sepharose 6B gel filtration chromatography. In 0.1% Triton X-100, the binding complex has a Stokes radius of 70 A, a sedimentation coefficient of 6.9 S, and a partial specific volume of 0.698 ml/g. The detergent-protein complex has a calculated molecular mass of 230 kDa. The estimated frictional ratio is 1.5. The native binding complex appears to consist of a dimer of identical subunits. The function of this ubiquitous protein remains unclear.     

Purification of the 5-hydroxytryptamine 5-HT3 receptor from NCB20 cells

A 5-hydroxytryptamine 5-HT3 receptor binding site has been purified from deoxycholate-solubilized NCB20 cell membranes. Purification (1,700-fold) was achieved in one step by affinity chromatography with L-685,603 immobilized on agarose. The 5-HT3 selective antagonist [3H]Q ICS 205-930 labeled a single population of receptors in the affinity-purified preparation with a Bmax of 3.1 +/- 0.9 nmol/mg protein and Kd of 0.40 +/- 0.05 nM (mean +/- S.E., n = 3). The rank order of potency for a series of competing compounds confirmed that [3H]Q ICS 205,930 was labeling a 5-HT3 receptor in the purified preparation, and the inhibition constants for all antagonists were unchanged after purification. The purified 5-HT3 binding site eluted from a Sepharose 6B gel filtration column in a similar manner to the crude solubilized preparation (Stokes radius of 4.9 nm, apparent molecular size 250,000). Polyacrylamide gel electrophoresis of the affinity-purified receptor showed two broad bands by silver staining, migrating with apparent molecular masses of 54,000 and 38,000. Gel filtration of the affinity purified material yielded a single peak labeled by [3H]Q ICS 205-930 with an apparent molecular size of 250,000, which was also composed of two bands of 54,000 and 38,000, consistent with these being the constituents of the 5-HT3 receptor.     

The low affinity binding site for the cocaine analog, WIN 35,428 is an artifact of freezing caudate tissue.

Binding of the cocaine analog [3H] WIN 35,428 was investigated in rat and rabbit caudate. In membranes prepared from fresh tissue, [3H] WIN 35,428 binding was characterized by a single high affinity site with a Kd of 2.5 nM for the rabbit and 5.3 nM for the rat. In contrast, [3H] WIN 35,428 binding to membranes prepared from frozen tissue (stored at -70 degrees C) revealed two binding sites, a high affinity site similar to the one observed in membranes from fresh tissue and a low affinity site with a Kd of 39 nM for the rabbit and 65 nM for the rat. The low affinity WIN 35,428 binding site was observed only in membranes derived from frozen tissue, suggesting that it was an artifact produced by the freezing/thawing process.     

Photoaffinity labeling of the tetrodotoxin binding component ofElectrophorus electricus electroplax

Radioactive azide derivatives of tetrodotoxin (TTX) were synthesized using 2-nitro-4-azidephenyl-[3H]beta alanine for the purpose of photolabeling of the Na channel. Three azide derivatives, N1, N2 and N3, were separated by ion exchange chromatography on Bio-Rex 70 resin and reversed phase high performance liquid chromatography. N3 was more stable and obtained at a higher yield than the other two derivatives. Bioactivity of N3 was one-twentieth of that of TTX. N3 showed reversible binding to membranes of Electrophorus electricus electroplax in the dark with Kd = 30 nM and B max = 5.2 pmol/mg protein. By photoirradiation, irreversible binding of N3 to the membranes was observed. A N3 binding component was solubilized by lubrol PX and partially purified from the electroplax membranes by Sephadex G25 and Sepharose 6B column chromatography. The component, purified 500 fold from the starting membranes, showed molecular weight of 10,000.     

Binding sites for inositol trisphosphate in the bovine adrenal cortex

Binding sites for inositol trisphosphate (IP3) have been identified in bovine adrenal cortex, employing [32P]IP3 prepared from human erythrocytes radiolabeled with [32P]ATP. IP3 was bound to adrenal microsomes with high affinity (Kd = 5 nM) and low capacity (186 fmol/mg protein). During kinetic studies, half-maximal binding was reached in less than one min at 4 degrees C, and dissociation was even more rapid with t1/2 of about 10 sec. [32P]IP2 showed no binding to the microsomal sites, which represent putative receptors at which IP3 acts to elevate intracellular calcium concentration during the actions of peptide hormones such as angiotensin II.     

Characterization of gingival epithelium epidermal growth factor receptor

The binding characteristics of gingival epithelium epidermal growth factor (EGF) receptor were investigated using epithelial cell membranes from bovine gingiva. The binding of [125I]EGF was found to be time and protein concentration dependent, reversible, and specific. Unlabeled EGF competed for [125I]EGF binding with IC50 of 0.25nM and maximum displacement of 93% at 0.81nM. Scatchard analysis of the binding data inferred the presence of two binding sites, one of high affinity (Kd = 3.3 nM and Bmax = 47.3fmol/mg protein) and the other of a low affinity (Kd = 1.6 microM and Bmax = 1.9pmol/mg protein). Crosslinking of [125I]EGF to gingival membranes followed by polyacrylamide gel electrophoresis and autoradiography revealed a receptor protein of 170kDa.     

Subcellular localization and some properties of lipoxygenase activity in human blood platelets.

Lipoxygenase activity was measured in human platelet subcellular fractions. From a sonicated platelet preparation, a granule fraction, mixed membranes (surface and intracellular) and cytosol fractions were separated by differential centrifugation. With respect to activities in the sonicated preparation, the lipoxygenase was slightly enriched in both the cytosol and mixed-membrane fractions and consistently de-enriched in the granule fractions. Approx. 65% and 20% of the total cell enzyme activity were found in the cytosol and mixed membranes respectively, with only 8% present in the granule fraction. Additionally we measured the lipoxygenase activity in purified surface- and intracellular-membrane subfractions prepared from the mixed membranes by free-flow electrophoresis. There was a slight enrichment in activity in the intracellular membrane fraction compared with that in the mixed membranes, and a depletion of activity in the surface membranes. Characterization of the enzyme activity, i.e. time course, pH-dependence, Ca2+-dependence, Vmax. and Km for arachidonic acid, and the carbon-position specificity for this acid, failed to reveal any significant differences between the membrane-bound and soluble forms of the lipoxygenase. These findings suggest that in human platelets the same lipoxygenase is associated with the membranes as in the cytosol and that the membrane-bound activity predominates in intracellular membrane elements.     

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

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

Formation of diacyl- and alkylacylphosphatidylcholine by the membranes of human platelets

We have investigated the distribution and fatty acid preference of two acyl-CoA transferase activities in a human platelet mixed membrane fraction and in well-characterised surface and intracellular membrane subfractions prepared from it by high-voltage free-flow electrophoresis. One transferase inserts long-chain unsaturated fatty acids into 1-acyllysophosphatidylcholine (1-acyl-LPC) and the other into lyso-platelet-activating factor (LPAF). Both transferase activities were approx. 4-fold enriched in the intracellular membranes with respect to their specific activities in the mixed membranes. The surface membrane activities were correspondingly depleted. Using 1-acyl-LPC as the acceptor, all the intracellular membrane preparations showed transferase preference for the CoA ester of 8,11,14-eicosatrienoic acid. In contrast when LPAF was the acceptor the CoA esters of linoleic and arachidonic acid were the preferred donors.     

Different localization of inositol 1,4,5-trisphosphate and ryanodine binding sites in rat liver.

The distribution of inositol 1,4,5-trisphosphate and ryanodine binding sites between plasma membrane, microsomal, and mitochondrial fractions of rat liver were compared. IP3 bound mostly to the plasma membrane fraction (Kd = 6 nM; Bmax = 802 fmol/mg protein). Some IP3 binding sites were also present in the microsomal and mitochondrial fractions (Kd = 2.5 and 2.9 nM; Bmax = 35 and 23 fmol/mg protein respectively). The possibility that these binding sites are due to contamination of the fractions with plasma membrane cannot be excluded. Binding of IP3 to the plasma membrane was inhibited by heparin but not by either caffeine or tetracaine. High-affinity ryanodine binding sites were present mostly in the microsomal fraction (Kd = 13 nM; Bmax = 301 fmol/mg protein). Lower affinity binding sites were also found to be present in the mitochondrial and plasma membrane fractions. Binding of ryanodine to the microsomal fraction was inhibited by both caffeine and tetracaine but not by heparin. These data demonstrate that IP3 and ryanodine binding sites are present in different cellular compartments in the liver. These differences in the localization of the binding sites might be indicative of their functional differences.     

Adrenergic receptors in the liver parenchyma in children with chronic hepatitis]

In the present study adrenergic receptors have been investigated in liver parenchyma, obtained at the resection of extrahepatic portal hypertension children without parenchymal affection (control group, n-7) and the resection of children in parenchymal affection (group of chronic hepatitis children, n-6). It has been shown, that the binding of beta-adrenergic radioligand 3H-dihydroalprenolol (3H-DHA) in liver parenchyma membranes of both control and chronic hepatitis groups was saturable and showed high affinity. The Scatchard analysis of the binding data indicated that the binding site was characterized by Kd and Bmax of 1.2 +/- 0.5 nM, 261.2 +/- 50 fmol/mg, respectively, for the control group; and 0.9 +/- 0.15 nM, 68.5 +/- 18.8 fmol/mg, respectively, for the group of chronic hepatitis patients; (mean+SEM). The binding of alpha 1-adrenergic antagonist 3H-prazosin (3H-PRZ) in liver parenchyma was also saturable and showed high affinity. The binding site is characterized by Kd = 0.6 +/- 0.12 nM, Bmax = 92.8 +/- 8.0 fmol/mg, for the control group; and Kd = 0.8 +/- 0.15 nM, Bmax = 195.0 +/- 22.0 fmol/mg, for the group of chronic hepatitis. It has been found that the number of binding sites of 3H-DHA significantly decreased and the number of binding sites of 3H-PRZ did not change in chronic hepatitis liver parenchyma in comparison with the control group. The results obtained suggest the important role of beta-adrenergic receptors in the pathogenesis of chronic hepatitis and in liver regeneration in children.     

Characterization of specific binding sites for corticosterone in mouse liver plasma membrane

The specific binding of [3H]corticosterone to mouse liver purified plasma membrane fractions is a saturable, reversible, and temperature-dependent process. Only one type of independent and equivalent binding sites has been determined in plasma membrane (Kd = 4.1 nM and Bmax = 3368 fmol/mg). As can be deduced from displacement data obtained in plasma membrane, the high-affinity binding site is different from nuclear glucocorticoid, nuclear progesterone, and Na+, K(+)-ATPase digitalis receptors. Probably this corticosterone binding site or receptor is the same one determined previously for [3H]cortisol in mouse liver plasma membrane. Such beta- and alpha-adrenergic antagonists as propranolol and phentolamine did not affect [3H]corticosterone binding to plasma membranes; therefore, this binding site is independent of these receptors. The binding sites in plasma membranes are not exclusive for corticosterone, but other steroids are also bound with very different affinities.     

[3H]Muscimol Binding Site on Purified Benzodiazepine Receptor

The presence of a [3H]muscimol binding site on the purified benzodiazepine receptor was demonstrated. The purified protein was apparently homogeneous as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis (stained with silver), with a molecular weight of 60,000 +/- 3000. The benzodiazepine binding sites were characterized as being of the central type and the [3H]flunitrazepam binding was enhanced by GABA. This activation was antagonized by bicuculline. [3H]Muscimol specifically binds to the benzodiazepine receptor. The Scatchard plot indicates a Kd of 23 nM and the ratio [3H]flunitrazepam/[3H]muscimol is approximately unity.     

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.