Purification of a histamine H3 receptor negatively coupled to phosphoinositide turnover in the human gastric cell line HGT1.

The histamine H3 receptor agonist (R)alpha-methylhistamine (MeHA) inhibited, in a nanomolar range, basal and carbachol-stimulated inositol phosphate formation in the human gastric tumoral cell line HGT1-clone 6. The inhibition was reversed by micromolar concentrations of the histamine H3 receptor antagonist thioperamide and was sensitive to cholera or pertussis toxin treatment. Using [3H]N alpha-MeHA as specific tracer, high affinity binding sites were demonstrated with a Bmax of 54 +/- 3 fmol/mg of protein and a KD of either 0.61 +/- 0.04 or 2.2 +/- 0.4 nM, in the absence or presence of 50 microM GTP[gamma]S, respectively. The binding sites were solubilized by Triton X-100 and prepurified by gel chromatography. They were separated from the histamine H2 receptor sites by filtration through Sepharose-famotidine and finally retained on Sepharose-thioperamide. The purified sites concentrated in one single silver-stained protein band of 70 kDa in SDS-polyacrylamide gel electrophoresis. They specifically bound [3H]N alpha-MeHA with a KD of 1.6 +/- 0.1 nM and a Bmax of 12,000 +/- 750 pmol/mg of protein. This corresponds to a 90,225-fold purification over cell lysate and a purity degree of 84%. Binding was competitively displaced by N alpha-MeHA (IC50 = 5.8 +/- 0.7 nM), (R) alpha-MeHA (IC50 = 9 +/- 1 nM), and thioperamide (IC50 = 85 +/- 10 nM), but not by famotidine (H2 antagonist) or by mepyramine (H1 antagonist). These findings provide the first evidence for solubilization, purification, and molecular mass characterization of the histamine H3 receptor protein and for the negative coupling of this receptor phosphatidylinositol turnover through a so far unidentified G protein.     

Purification and ligand-receptor analysis of insulin-related peptides from pedal ganglion of the mollusc Anodonta cygnea

Six insulin-related peptides (IRPs) from pedal ganglions of the molluscs Anodonta cygnea have been isolated and purified by reverse-phase chromatography. Each peptide (designated as IRP8-IRP13) showed its own retention time on the HPLC column. The testing of IRPs in radioreceptor systems specific for insulin and insulin growth factor-I (IGF-I) showed their ability to bind to both types of receptors. The concentration of IRPs, producing a 50% inhibition of porcine 125I-insulin binding with rat liver plasma membrane receptors (IC50) for IRP 10, was 1167 nM, IRP11--833 nM, IRP13--1333 nm. IRP8, IRP9, IRP12 in the maximum concentration of 10(4) ng/ml displaced less than 50% of labeled hormone. All of the six peptides were capable of competing with human 125I-IGF-I for binding to receptors of a fraction of rat brain membranes. IRP8, IRP9 and IRP12 had close means equal to 1167 nM, 1500 nM, 1167 nM, respectively. Another group including IRP10, IRP11 and IRP13 showed a much higher activity (833, 83 and 500 nM, respectively). The results obtained from radioligand analysis revealed the predominance of IGF-I binding properties in all peptides of pedal ganglions. At the same time, apparent proximity of IRP's physico-chemical characteristics to porcine insulin, and also the revealed dose-dependent binding to both insulin and IGF-I receptors suggest a bifunctionality of mollusc peptides. The expression level of this bifunctionality may be associated with the molecular structure pecularities of individual isoforms.     

Solubilization of Calcium Channel Antagonist Binding Sites from Rat Brain

Calcium antagonist binding sites were solubilized from rat brain membranes using the detergent 3-[(3-cholamidopropyl)dimethylammonio] 1-propanesulfonate (CHAPS). CHAPS-solubilized [3H]nitrendipine binding sites are saturable over a range of 0.05-4 nM and Scatchard analysis reveals a single, high-affinity (KD = 0.49 +/- 0.10 nM), low-capacity (Bmax = 56 +/- 4 fmol/mg of protein) binding site. Reversible ligand competition experiments using solubilized binding sites demonstrated appropriate pharmacologic specificity, with dihydropyridines (nifedipine = nitrendipine greater than Bay K 8644) completely displacing binding, verapamil partially displacing binding, and diltiazem enhancing binding, as previously described in membrane preparations. Lyophilized Crotalus atrox venom was purified by ion exchange chromatography followed by gel filtration to a single peptide band on sodium dodecyl sulfatepolyacrylamide gel electrophoresis. This fraction of molecular weight 60,000 competitively inhibits [3H]nitrendipine binding to both membrane and soluble preparations with an IC50 of 5 micrograms/ml. This polypeptide should serve as a useful ligand for future efforts in purifying the dihydropyridine calcium channel binding site in brain.     

Urokinase binding and receptor identification in cultured endothelial cells.

Recombinant human single-chain urokinase (rscu-PA), two-chain urokinase (tcu-PA), and diisopropyl-fluorophosphate-treated tcu-PA (DFP-tcu-PA) bound to cultured human and porcine endothelial cells in a rapid, saturable, dose-dependent and reversible manner. Analysis of specific binding results in cultured human umbilical vein endothelial cells (HUVECs) gave the following estimated values for Kd and Bmax: 0.57 +/- 0.08 nM (mean +/- S.E.) and 188,000 +/- 18,000 sites/cell for 125I-labeled rscu-PA; 0.54 +/- 0.10 nM and 132,000 +/- 23,900 sites/cells for 125I-labeled tcu-PA; 0.89 +/- 0.14 nM and 143,000 +/- 30,300 sites/cell for 125I-labeled DFP-tcu-PA, respectively. Values for Kd were similar for primary and subcultured (six passages) HUVECs, but Bmax values were lower in subcultured HUVECs. Similar Kd values were found in cultured porcine endothelial cells; however, Bmax values varied depending on the endothelial cell type. All 125I-labeled urokinase forms yielded similar cross-linked approximately 110-kDa ligand-receptor complexes with cultured HUVECs, and 125I-labeled DFP-tcu-PA bound to a single major approximately 55-kDa protein in whole-cell lysates (ligand blotting/autoradiography), suggesting the presence of a single major approximately 55-kDa urokinase receptor in cultured HUVECs. The approximately 55-kDa urokinase receptor, isolated from several separate batches of cultured HUVECs (3-5 micrograms of protein, approximately 1 x 10(9) cells), by ligand affinity chromatography, exhibited the following properties: retained biologic activity as evidenced by its ability to bind 125I-labeled rscu-PA by ligand blotting/autoradiography and formation of a cross-linked 125I-labeled approximately 110-kDa rscu-PA-receptor complex; single-chain approximately 55-kDa protein, following reduction; complete conversion to and formation of a single major deglycosylated approximately 35-kDa protein, following treatment with N-glycanase.     

Isolation and cDNA cloning of a novel galanin-like peptide (GALP) from porcine hypothalamus

Galanin is a widely distributed neuropeptide with a variety of physiological functions. Three galanin receptor subtypes, GALR1, GALR2, and GALR3, have been reported. We isolated a novel galanin-like peptide (GALP) from porcine hypothalamus by observing its activity for increasing [(35)S]GTPgammaS binding to a membrane preparation of GALR2-transfected cells. The peptide had 60 amino acid residues and a non-amidated C terminus. The amino acid sequence of GALP-(9-21) was completely identical to that of galanin-(1-13). A cloned porcine GALP cDNA indicated that GALP was processed from a 120-amino acid GALP precursor protein. The structures of rat and human GALP-(1-60) were deduced from cloned cDNA, which indicated that the amino acid sequences 1-24 and 41-53 were highly conserved between humans, rats, and pigs. Receptor binding studies revealed that porcine GALP-(1-60) had a high affinity for the GALR2 receptor (IC(50) = 0.24 nM) and a lower affinity for the GALR1 receptor (IC(50) = 4.3 nM). In contrast, galanin showed high affinity for the GALR1 (IC(50) = 0.097 nM) and GALR2 receptors (IC(50) = 0.48 nM). GALP is therefore an endogenous ligand that preferentially binds the GALR2 receptor, whereas galanin is relatively non-selective.     

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.     

Comparison of the biological activity of canine and porcine motilin in rabbit

The biological activity of porcine and canine motilin was studied in rabbits by establishing dose-response curves of both peptides using two different methods. The dissociation constant, obtained from the displacement of iodinated porcine motilin by canine motilin was 0.6 +/- 0.3 nM, versus 1.2 +/- 0.4 nM for porcine motilin. For the 13-norleucine and 13-leucine analogues of porcine motilin a value of 0.8 +/- 0.3 nM was obtained. Both motilins were almost equipotent in stimulating the in vitro contractile response of longitudinal smooth muscle strips: half-maximal effect was achieved at a concentration of 1.0 +/- 0.1 nM for canine versus 1.3 +/- 0.2 nM for the 13-norleucine analogue of porcine motilin. We conclude that porcine and canine motilin have a comparable bioactivity in the rabbit, although canine motilin is slightly more effective. The motilin receptor is probably specific for the N-terminal portion which is identical in both molecules.     

Interaction of thrombospondin with resting and stimulated human platelets

The interaction of isolated and radioiodinated thrombospondin with washed human platelets has been characterized. The ligand bound to nonstimulated and thrombin-stimulated platelets in a time-dependent manner, and apparent steady state was reached within 25 min. Binding was not due to iodination of the ligand and was inhibited by nonlabeled thrombospondin but not by unrelated proteins, and bound ligand was identical with thrombospondin in terms of subunit structure. Nonlinear curve-fitting analyses of binding to resting platelets suggested the presence of a single class of sites which bound 3,100 +/- 1,000 molecules/platelet with an apparent Kd of 50 +/- 20 nM. This interaction was not attributable to contaminating cells or inadvertant platelet activation. Binding to thrombin-stimulated platelets had a lower apparent affinity (Kd = 250 +/- 100 nM) and higher apparent capacity (35,600 +/- 9,600 molecules/platelet). Thrombin-enhanced binding was dependent upon agonist dose and platelet stimulation. Fibrinogen, a monoclonal antibody to GPIIb-IIIa, temperature, and divalent ions had differential effects upon thrombospondin binding to resting and stimulated platelets, suggesting the presence of two distinct mechanisms of thrombospondin binding to platelets. While thrombospondin binding to thrombin-stimulated platelets occurs with characteristics similar to those observed for fibrinogen, fibronectin, and von Willebrand Factor, its high affinity interaction with resting platelets is unique to this adhesive glycoprotein.     

Molecular identification and structural requirement of vasoactive intestinal peptide (VIP) receptors in the human colon adenocarcinoma cell line, HT-29.

The human colon adenocarcinoma cell line HT-29 in culture exhibits a cyclic AMP production system highly sensitive to vasoactive intestinal peptide (VIP), making HT-29 cells a unique cultured cell system for studying the mechanism of VIP action [Laburthe, Rousset, Boissard, Chevalier, Zweibaum & Rosselin (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2772-2775]. The quantitative characteristics of VIP receptors in HT-29 cells and their structural requirement and molecular size were studied. 125I-labeled VIP bound in a time-dependent manner to HT-29 cell homogenates. At equilibrium (60 min incubation at 30 degrees C), unlabelled VIP in the 0.01-10 nM concentration range competed with 125I-VIP for binding to cell homogenates. Scatchard analysis of binding data gave a straight line, indicating that VIP bound to a single population of sites with a KD of 0.12 +/- 0.02 nM and a capacity of 120 +/- 9 fmol/mg of protein. The structural requirement of these receptors was studied with peptides structurally related to VIP, either natural or synthetic. Several peptides inhibited 125I-VIP binding to HT-29 cell homogenates with the following order of potency, which is typical of the human VIP receptor: VIP (IC50 = 0.1 nM) greater than VIP-(2-28)-peptide (IC50 = 13 nM) greater than human growth hormone releasing factor (IC50 = 56 nM) greater than peptide histidine isoleucine amide (IC50 = 80 nM) greater than secretin (IC50 greater than 10 000 nM). To characterize the molecular component(s) of the VIP receptor in HT-29 cells, 125I-VIP was covalently bound to cell homogenates by using the cross-linker dithiobis(succinimidyl propionate). Sodium dodecyl sulphate/polyacrylamide-gel autoradiographic studies of affinity-labelled cell homogenates revealed two major bands, corresponding to 125I-VIP-protein complexes of Mr 66 000 and 16 000. The labelling of the Mr-66 000 component was specific, since it was abolished by native VIP, whereas that of the Mr-16 000 component was not. Densitometric scanning of autoradiographs indicated that the labelling of the Mr-66 000 complex was inhibited by low VIP concentrations in the 0.1-10 nM range (IC50 = 0.6 nM), but was unaffected by 1 microM-glucagon or octapeptide of cholecystokinin. It was also decreased by VIP-(2-28)-peptide with a potency 1% that of VIP. Assuming that one molecule of 125I-VIP bound per molecule of protein, one protein of Mr 63 000 was identified as a component of the VIP receptor in HT-29 cells.     

Proteolytic excision and in situ cyclization of a bioactive loop from an REI-VL presentation scaffold.

Covalent cyclization of peptides is an important tool in structure-function analysis of bioactive peptides, because it constrains the molecule to enrich or exclude the receptor-bound conformation. Previously we described a 2-step procedure for cyclizing purified, native peptides in aqueous solution by reacting a Met or Lys side chain with an iodoacetylated N-terminus (Wood SJ, Wetzel R, 1992a, Int J Pept Protein Res 39:533-539). We show here that the cyclization reaction scheme can be extended to peptides excised from proteins by endo-LysC proteolysis, which generates fragments terminating with Lys. To illustrate the method, we used an immunoglobulin VL domain (REI-VL) with an RGD-containing sequence engineered into its CDR3 and flanked by Lys residues. This REI-VL/RGD hybrid displayed an IC50 of 24 nM for ligand competition at the platelet fibrinogen receptor alpha IIb beta 3. The RGD-containing peptide excised by endo-LysC from the REI-VL presentation scaffold exhibited an IC50 of about 50 nM, and the corresponding cyclized peptide, and IC50 of about 10 nM. Significantly, both the N alpha-acylation and the cyclization reactions occur efficiently even in the context of the other endo-LysC fragments of REI-VL, which suggests that the reaction may prove useful in converting mixtures of endo-LysC products of many proteins into the corresponding cyclic peptides in situ.     

Identity of a membrane-bound vasoactive intestinal peptide-binding protein with calmodulin.

A vasoactive intestinal peptide (VIP)-binding protein purified from guinea pig lung membranes (p18) was digested with trypsin, and the amino acid sequence of the peptide fragments was determined. The sequence of six tryptic fragments of p18 was identical with subsequences present in mammalian calmodulin. Authentic porcine brain calmodulin and p18 co-migrated on an sodium dodecyl sulfate-electrophoresis gel and displayed identical chromatographic behavior on a reverse phase high performance liquid chromatography column. The VIP-binding properties of p18 and calmodulin were indistinguishable. Both proteins displayed saturable and apparent high affinity binding of VIP, evidenced by potent inhibition of complexation with [Tyr10-125I]VIP by unlabeled VIP (IC50 = 6.0-8.1 nM). Rat growth hormone releasing factor and a C terminally extended form of VIP ([Leu17]VIP-GKR) also displayed potent inhibition of the binding (IC50 = 6.4 and 4 nM, respectively). These neuropeptides are potential modulators of calmodulin function.     

Purification and properties of Arabidopsis thaliana type 1 protein phosphatase (PP1).

The Arabidopsis thaliana type 1 protein phosphatase (PP1) catalytic subunit was released from its endogenous regulatory subunits by ethanol precipitation and purified by anion exchange and microcystin affinity chromatography. The enzyme was identified by MALDI-TOF mass spectrometry from a tryptic digest of the purified protein as a mixture of PP1 isoforms (TOPP 1-6) indicating that at least 4-6 of the eight known PP1 proteins are expressed in sufficient quantities for purification from A. thaliana suspension cells. The enzyme had a final specific activity of 8950 mU/mg using glycogen phosphorylase a as substrate, had a subunit molecular mass of 35 kDa as determined by SDS-PAGE and behaved as a monomeric protein of approx. 39 kDa on Superose 12 gel filtration chromatography. Similar to the mammalian type 1 protein phosphatases, the A. thaliana enzyme was potently inhibited by Inhibitor-2 (IC(50)=0.65 nM), tautomycin (IC(50)=0.06 nM), microcystin-LR (IC(50)=0.01 nM), nodularin (IC(50)=0.035 nM), calyculin A (IC(50)=0.09 nM), okadaic acid (IC(50)=20 nM) and cantharidin (IC(50)=60 nM). The enzyme was also inhibited by fostriecin (IC(50)=22 microM), NaF (IC(50)=2.1 mM), Pi (IC(50)=9.5 mM), and PPi (IC(50)=0.07 mM). Purification of the free catalytic subunit allowed it to be used to probe protein phosphatase holoenzyme complexes that were enriched on Q-Sepharose and a microcystin-Sepharose affinity matrix and confirmed several proteins to be PP1 targeting subunits.     

Expression and characterization of rat soluble catechol-O-methyltransferase fusion protein

Rat soluble catechol-O-methyltransferase cDNA was cloned into the pCAL-n-FLAG vector and expressed in Escherichia coli as a fusion protein with a calmodulin-binding peptide tag. The recombinant protein, comprising up to 30% of the total protein in the soluble fraction of E. coli, was purified by calmodulin affinity chromatography and gel filtration. Up to 16 mg of pure recombinant enzyme was recovered per liter of culture. Recombinant catechol-O-methyltransferase, in the bacterial soluble fraction, exhibited the same affinity for adrenaline as rat liver soluble catechol-O-methyltransferase (K(m) 428 [246, 609] microM and 531 [330, 732] microM, respectively), as well as the same affinity for the methyl donor, S-adenosyl-l-methionine (K(m) 27 [9, 45] microM and 38 [21, 55] microM, respectively). In addition, both the recombinant and the liver enzymes displayed the same sensitivity to the inhibitor 3,5-dinitrocatechol (IC(50) 132 [44, 397] nM and 74 [38, 143] nM, respectively), and both had the same catalytic number, respectively, 10.1 +/- 1.5 min(-1) and 8.3 +/- 0.3 min(-1). The purified recombinant enzyme also displayed the same affinity for the substrate as the purified rat liver catechol-O-methyltransferase (K(m) 336 [75, 597] microM and 439 [168, 711] microM, respectively) as well as the same inhibitor sensitivity (IC(50) 44 [19, 101] nM and 61 [33, 111] nM, respectively). This recombinant form of catechol-O-methyltransferase is kinetically identical to the rat liver enzyme. This system provides an easy and quick way of obtaining large amounts of soluble catechol-O-methyltransferase for both pharmacological and structural studies.     

Synthesis and biological evaluation of halogenated naphthyridone carboxamides as potential ligands for in vivo imaging studies of substance P receptors

With the aim of developing new radioligands for in vivo studies of substance P receptors using positron emission tomography or single photon emission computed tomography, 2- and 3-halo naphthyridone-6-carboxamide derivatives were synthesized. Their affinities toward the target receptors were evaluated on CHO cells and compared to the unsubstituted analogue EP 00652218 (IC(50) = 100 nM +/- 20). The IC(50) value was not altered in the case of 2-chloro compound 1 (IC(50) = 100 nM +/- 15) and only slightly reduced for the 2-fluoro and -iodo analogues 6 and 8 (IC(50) = 500 nM +/- 80). A drastic reduction in binding (IC(50) > 1000 nM) was observed for the halogenated compounds 2-5, 7, and 9.     

Agonist-Dependent Internalization of γ-Aminobutyric AcidA/Benzodiazepine Receptors in Chick Cortical Neurons

An impermeant benzodiazepine receptor ligand was prepared by derivatization of the aminobenzodiazepine 1012-S with 4-sulfophenylisothiocyanate. The resulting N-(4-sulfophenyl)-thiocarbamoyl derivative of 1012-S (SPTC-1012S) was purified by reverse-phase HPLC, and the predicted structure was verified by mass spectrometry. The apparent affinity of SPTC-1012S (IC50 = 9.8 +/- 2.9 nM) for displacement of [3H]flunitrazepam from intact chick cortical neurons was similar to that of 1012-S (IC50 = 4.0 +/- 0.3 nM). However, at concentrations from 0.1 to 10 microM, 1012-S was consistently more efficacious than SPTC-1012S, a finding indicating that 6-8% of the benzodiazepine receptor pool was not accessible to the impermeant compound. This inaccessible pool was eliminated by permeabilization of the cells with saponin or Triton X-100, a result suggesting that approximately 7% of neuronal benzodiazepine receptors are intracellular. Acute treatment (1-4 h at 37 degrees C) of neurons with 100 microM gamma-aminobutyric acid (GABA) or 100 nM clonazepam had little effect on the level of [3H]flunitrazepam binding but increased the proportion of intracellular receptors by 61 and 74%, respectively, compared with untreated controls. Similar treatment with 1 mM GABA increased the level of intracellular sites by 154-176%. The effect of GABA on receptor internalization was blocked by cotreatment with the GABAA receptor antagonist R 5135. The results suggest that SPTC-1012S can be used as a probe to study the internalization of the GABAA/benzodiazepine receptor complex under normal conditions or following acute or chronic treatment with agonists.     

Activity of alpha- and theta-defensins against primary isolates of HIV-1

Theta-defensins are lectin-like, cyclic octadecapeptides found in the leukocytes of nonhuman primates. They are also homologues of the more familiar alpha-defensins expressed by humans and certain other mammals. This study compares the ability of six theta-defensins (hominid retrocyclins 1-3 and rhesus theta-defensins 1-3) and four human alpha-defensins (human neutrophil peptides (HNPs) 1-4) to bind gp120 and CD4. In addition, we compared the ability of these theta-defensins and HNP-1 to protect J53-BL cells (an indicator cell line) from primary HIV-1 isolates that varied in subtype and coreceptor usage. The most potent theta-defensin, retrocyclin-2, bound with exceptionally high affinity to gp120 (K(D), 9.4 nM) and CD4 (K(D), 6.87 nM), and its effectiveness against subtype B isolates (IC(50), 1.05 +/- 0.28 microg/ml; 520 +/- 139 nM) was approximately twice as great as that of HNP-1 on a molar basis. We also show, for the first time, that human alpha-defensins, HNPs 1-3, are lectins that bind with relatively high affinity to gp120 (K(D) range, 15.8-52.8 nM) and CD4 (K(D) range, 8.0-34.9 nM). Proteins found in human and FBS bound exogenous HNP-2 and retrocyclin-1, and competed with their ability to bind gp120. However, even the low concentrations of alpha-defensins found in normal human serum suffice to bind over half of the gp120 spikes on HIV-1 and a higher percentage of cell surface CD4 molecules. Although this report principally concerns the relationship between carbohydrate-binding and the antiviral properties of alpha- and theta-defensins, the lectin-like behavior of defensins may contribute to many other activities of these multifunctional peptides.     

Synthesis and molecular characterization of a biotinylated analog of [Lys]bradykinin.

We report the synthesis and molecular characterization of a biotinylated analog of kallidin, [Lys]bradykinin. Bradykinin was prepared by solid phase peptide synthesis. Before cleavage from the resin, a biotin moiety was coupled to the epsilon amino group of a lysine in the zeroth position of the bradykinin peptide. An omega-amino caproic acid spacer was incorporated between the biotin group and the N-terminal lysine. The biotinylated peptide was deprotected, cleaved from the resin and purified by RP-HPLC. The identity of this analog was confirmed by amino acid analysis and FAB-mass spectrometry. Biotinyl [Lys]bradykinin (BLBK, mol, wt. = 1528) inhibited [3H]-bradykinin binding to guinea pig ileum homogenates dose dependently, with an IC50 of 28.9 +/- 6 nM. The IC50 for [Lys]bradykinin was approximately 10-fold lower, 3.2 +/- 0.6 nM. BLBK induced contractility in an isolated guinea pig smooth muscle preparation with an EC50 of 129 +/- 14 nM; the corresponding value for [Lys]bradykinin was 29 +/- 8 nM. These data are consistent with the difference in binding potency observed for BLBK compared to [Lys]bradykinin. In an ELISA assay using BLBK and affinity-purified rabbit anti-bradykinin antibody, BLBK bound to anti-bradykinin antibody with an EC50 = 1.21 +/- 0.54 nM. Rank order potencies for several bradykinin peptide analogs suggest that the epitope on bradykinin recognized by the antibody is likely to be at the carboxy terminus of the peptide.     

Synthesis and structure-activity relationship of a novel, non-hydroxamate series of TNF-alpha converting enzyme inhibitors

A novel series of TNF-alpha converting enzyme (TACE) inhibitors which are non-hydroxamate have been discovered. These compounds use a triazolethione moiety as the zinc binding ligand and exhibit IC50 values from 1.5 to 100 nM in a porcine TACE assay. They also have excellent selectivities over other MMPs.     

Binding of Brain and Atrial Natriuretic Peptides to Cultured Mouse Astrocytes and Effect on Cyclic GMP

125I-Porcine brain natriuretic peptide (125I-pBNP) bound to mouse astrocytes in primary culture in a time-dependent manner (t1/2 = 4.5 min), similar to 125I-human atrial natriuretic peptide (125I-hANP) (t1/2 = 5 min). Binding was saturable and reached equilibrium after 90 min at 22 degrees C for both radioligands. Scatchard analysis suggested a single class of binding sites for pBNP with a binding affinity and capacity (KD = 0.08 nM; Bmax = 78.3 fmol/mg of protein) similar to those of hANP1-28 (KD = 0.1 nM; Bmax = 90.3 fmol/mg of protein). In competition binding studies, pBNP or human/rat atrial natriuretic peptide (ANP) analogues [hANP1-28, rat ANP1-28 (rANP1-28), and rANP5-28] displaced 125I-hANP, 125I-pBNP, and 125I-rANP1-28 completely, all with IC50 values of less than nM (0.14-0.83 nM). All four peptides maximally stimulated cyclic GMP (cGMP) production by 10 min at 22 degrees C at concentrations of 1 microM with EC50 values ranging from 50 to 100 nM. However, maximal cGMP induction by brain natriuretic peptide (BNP) (25.9 +/- 2.1 pmol/mg of protein) was significantly greater than that by hANP1-28 (11.5 +/- 2.2 pmol/mg of protein), rANP1-28 (16.5 +/- 2.0 pmol/mg of protein), and rANP5-28 (15.8 +/- 2.2 pmol/mg of protein). These studies indicate that BNP and ANPs act on the same binding sites and with similar affinities in cultured mouse astrocytes. BNP, however, exerts a greater effect on cGMP production. The difference in both affinity and selectivity between binding and cGMP production may indicate the existence of receptor subtypes that respond differentially to natriuretic peptides despite similar binding characteristics.