Elucidation of structural requirements of mastoparan for mast cell activation-toward the comprehensive prediction of cryptides acting on mast cells.

Mastoparan, a toxic peptide from wasp venom, induces various biological functions including histamine release from rat peritoneal mast cells. Here we report that, for the activation of mast cells by mastoparan, at least two positively charged side chains are required on the hydrophilic side of the amphiphilic structure of the peptide. The present results are expected to be utilized for the bioinformatic and comprehensive identification of endogenous mast cell-stimulating cryptides.

Stimulation of calcium mobilization but not proliferation by bombesin and tachykinin neuropeptides in human small cell lung cancer cells.

The tachykinin family of neuropeptides, including substance P and neurokinins A and B, induce a transient increase in intracellular free calcium concentration in human small cell lung carcinoma (SCLC) cells, as measured with a calcium indicator fura-2. The effects are dose dependent and even greater than that of bombesin at equimolar concentrations in these cells. The tachykinins, like bombesin, induce calcium mobilization mainly from intracellular store(s). None of the peptides, however, shows a stimulatory effect on DNA synthesis. In addition, exogenously applied bombesin does not stimulate DNA synthesis at any concentration tested. We also examined the effects of a recently reported bombesin antagonist [D-Arg1, D-Phe5, D-Trp7,9, Leu11]substance P in SCLC cells, and compared them to those in Swiss 3T3 fibroblasts in which the mitogenic effect of bombesin is well characterized. The antagonist at 10(-5) M completely abolishes the Ca2+-mobilizing effect of 10(-7) M bombesin in SCLC cells, and that of 10(-9) M but not 10(-7) M bombesin in Swiss 3T3 cells. The antagonist at this concentration effectively inhibits the mitogenic action of bombesin (10(-9) M) in Swiss 3T3 cells; however, much higher doses (approximately 10(-4) M) are needed to inhibit DNA synthesis in SCLC cells. Moreover, the antagonist inhibits DNA synthesis in bombesin/gastrin-releasing peptide-nonproducing cells with a similar dose dependency as in producing cells. These results indicate that bombesin/gastrin-releasing peptide and other calcium mobilizing peptides do not always act as a growth factor in SCLC cells, and that the bombesin antagonist could inhibit growth of SCLC cells through a mechanism other than bombesin antagonism.

Isolation and characterization of Cox17p from porcine heart by determining its survival-promoting activity in NIH3T3 cells.

We have found that the gel filtration fraction of porcine heart extract clearly promoted the survival of NIH3T3 fibroblast cells in the serum-free medium condition. A structural analysis showed that the active fraction contained a novel peptide, porcine Cox17p (p-Cox17p), which was recently reported by Chen et al. as dopuin (Z. W. Chen et al., Eur. J. Biochem. 249 (1997) 518-522). Porcine Cox17p/dopuin possesses high sequence homology to the product of human COX17 gene (h-Cox17p). Although Cox17p has been implied to be involved in copper recruitment to mitochondria and in the functional assembly of cytochrome oxidase in yeast, its role in mammalian cells is unknown. In this study, we chemically synthesized p-Cox17p to investigate its biological effects. Refolding experiments of synthesized linear p-Cox17p revealed the existence of mostly one pattern of three intrachain disulfide bridges similar to that of native p-Cox17p, because the main oxidized p-Cox17p was completely co-eluted with the natural product. The addition of heavy metal ions such as copper, zinc and cadmium significantly inhibited the formation of the oxidized form, suggesting that reduced p-Cox17p may interact directly with these metal ions. The reduced and oxidized forms of p-Cox17p were also confirmed to promote the survival of NIH3T3 cells in serum-free medium as observed with the natural product, indicating that Cox17p may be a bioactive peptide.

Chemo-enzymatic synthesis of a bioactive peptide containing a glutamine-linked oligosaccharide and its characterization.

A bioactive peptide containing a glutamine-linked oligosaccharide was chemo-enzymatically synthesized by use of the solid-phase method of peptide synthesis and the transglycosylation activity of endo-beta-N-acetylglucosaminidase. Substance P, a neuropeptide, is an undecapeptide containing two L-glutamine residues. A substance P derivative with an N-acetyl-D-glucosamine residue attached to the fifth or sixth L-glutamine residue from the N-terminal region was chemically synthesized. A sialo complex-type oligosaccharide derived from a glycopeptide of hen egg yolk was added to the N-acetyl-D-glucosamine moiety of the substance P derivative using the transglycosylation activity of endo-beta-N-acetylglucosaminidase from Mucor hiemalis, and a substance P derivative with a sialo complex-type oligosaccharide attached to the L-glutamine residue was synthesized. This glycosylated substance P was biologically active, although the activity was rather low, and stable against peptidase digestion. The oligosaccharide moiety attached to the L-glutamine residue of the peptide was not liberated by peptide-N(4)-(N-acetyl-beta-D-glucosaminyl) asparagine amidase F.

Thermodynamics of a beta-hairpin structure: evidence for cooperative formation of folding nucleus.

To elucidate early nucleation stages in protein folding, multi-probed thermodynamic characterization was applied to the beta-hairpin structural formation of G-peptide, which is a C-terminal fragment of the B1 domain of streptococcal protein G. The segment corresponding to the sequence of G-peptide is believed to act as a nucleus during the folding process of the B1 domain. In spite of the broad thermal transition of G-peptide, nuclear magnetic resonance (NMR) melting measurements combined with our original analytical theory enabled us to obtain the thermodynamic properties of the beta-hairpin formation with considerable accuracy. Additionally, all the thermodynamic properties determined by every NMR probe on both the main-chain and the side-chains were quite similar, and also comparable to the values that were independently determined by calorimetric analysis of G-peptide. These results demonstrate that G-peptide folds cooperatively throughout the molecule. In other words, the formation of the beta-hairpin is interpreted as the fashion of a first-order phase transition between two states without any distinguishable intermediates. This cooperative formation of the short linear peptide consisting of only 16 residues provides insight into not only the first folding events of the B1 domain, but also the general principles of proteins in terms of structural hierarchy, stability and folding mechanism.

Determination of electrostatic potential around specific locations on the surface of actin by diffusion-enhanced fluorescence resonance energy transfer.

In this study we have established systematic procedures for the measurement of electrostatic potentials around specific and localized portions of protein surfaces. Diffusion-enhanced fluorescence energy transfer was used for these determinations. Energy transfer from donor molecules to freely diffusing acceptors is sensitive to the electrostatic potential around the donor when the acceptors possess electric charges. To quantify this sensitivity phenomenon in a controllable environment, we observed energy transfer from excited terbium chelate donors of known electric charge to a series of acceptors of different charges but bearing the same chromophore group. The rate of energy transfer was calculated theoretically (considering the structural arrangement of charged groups around the chromophore center), as well as determined experimentally (by time-resolved detection of terbium luminescence), with the two values obtained by the different means in close agreement. Having established the validity of this procedure using a relatively simple system, we studied the electrostatic conditions around two specific sites on the surface of actin molecules. A negative potential was found at both sites; the potential at one location (around the myosin binding site) was nearly neutralized by the addition of myosin subfragment-1, while the potential at the other site (around the phalloidin binding site) was not significantly affected.

Antigenic sites on the arginine-rich carboxyl-terminal domain of the capsid protein of hepatitis B virus distinct from hepatitis B core or e antigen.

The capsid protein of hepatitis B virus (P19) is made of 183 amino acids and carries the antigenic sites of hepatitis B core antigen (HBcAg) and hepatitis B e antigen (HBeAg) on the amino-terminal domain. The carboxyl-terminal domain of P19 (amino acids 150-183) is arginine-rich (47%) and faces the interior of the nucleocapsid for the binding with DNA. Monoclonal antibody was raised against an antigenic site on this protamine-like region of P19, which was distinct from HBcAg or HBeAg sites, and the novel antigenic site(s) was provisionally designated as hepatitis B inner core antigen (HBicAg). When P19 in a low concn (150 ng/ml) was immobilized on the solid surface, HBicAg sites were preserved, while HBcAg or HBcAg sites were no longer available on it. This allowed the detection of antibodies against HBicAg (anti-HBic), by sandwiching them between immobilized P19 and anti-IgG labeled with horseradish peroxidase. Anti-HBic was detected in sera from HBsAg carriers, typically those seropositive for antibody to HBeAg. A synthetic arginine-rich decapeptide, with a sequence of Arg-Arg-Arg-Gly-Arg-Ser-Pro-Arg-Arg-Arg, representing amino acids 150-159 of P19 and conserved in the majority of reported hepatitis B virus, absorbed the activity to bind with P19 in seven (44%) out of 16 sera containing anti-HBic. These results indicate that the decapeptide carries an HBicAg epitope and the remaining amino acid sequence of the arginine-rich carboxyl terminal domain (160-183) may be responsible for the other HBicAg epitopes.

Effects of substance P and substance P-(6-11) on hormone release from isolated perfused pancreas: their opposite actions on rat and canine islets.

The effects of substance P (SP) and SP-(6-11) (SP6-11) on hormone secretion from the isolated perfused pancreas were compared in rats and dogs under the same conditions. In the rat, SP inhibited insulin secretion in a dose-dependent manner in a concentration range of 0.1-10 nM. Glucagon secretion was inhibited at a minimal dose of 10 nM SP. No significant effect on somatostatin secretion was obtained. SP6-11 exhibited the identical inhibitory potency as SP on both insulin and glucagon release from the rat pancreas. In the canine pancreas, by contrast, 1 and 10 nM SP and SP6-11, respectively, potentiated the release of insulin, glucagon, and somatostatin. Potentiation by SP6-11 was less than that by SP. These results demonstrate species differences in the effects of SP and SP6-11 on the release of pancreatic hormones.

1H NMR study on the conformation of bacitracin A in aqueous solution.

The conformation of bacitracin A, a widely used cyclic dodecapeptide antibiotic in aqueous solution, has been investigated using 500 MHz 1H NMR and molecular modeling. Findings revealed that a region (residues 1-6) is folded over the cyclic ring, resulting in metal coordination sites, a thiazoline ring, and Glu4 and His10 being proximate to each other.

Complement assembly of two fragments of the streptococcal protein G B1 domain in aqueous solution.

We examined the complementation of various pairs of fragments derived from the streptococcal protein G B1 domain by NMR and CD. Most were not associated; however, one pair of fragments (1-40) and (41-56) interacted sufficiently enough to regenerated a stable 1:1 complex, Kd = 9 x 10(-6) M. A 2D-NMR analysis showed that the structure of the complex resembled that of native domain. Here we discuss the complementation from the viewpoint of the folding pathway of the protein.

Inhibitory effect of taurine on wet-dog shakes produced by [D-Ala2,Met5] enkephalinamide with reference to effects on hippocampal epileptic discharges.

The effects of taurine on wet-dog shakes produced by [D-Ala2,Met5]enkephalinamide (DAME) were investigated in rats. Wet-dog shakes and epileptic discharges in the hippocampus were produced by intraventricular administration of 50 micrograms of DAME. Pretreatment with 10 microliter of taurine, given intraventricularly in a dose of 0.95 mumol, inhibited wet-dog shakes and epileptic discharges in the hippocampus. While the same dose of gamma-aminobutyric acid (GABA) also inhibited the wet-dog shakes, the same dose of L-leucine did not suppress them. These observations indicate that the inhibition of DAME-induced wet-dog shakes by taurine is associated with the suppression of seizure activities in the hippocampus. The possibility that taurine possesses an antagonistic action on opioid peptides is discussed.

Stoichiometry of actin and phalloidin binding: one molecule of the toxin dominates two actin subunits.

Sonication of F-actin in the absence of added ATP in the solvent induces denaturation of the actin. When phalloidin is added to actin at the molar ratio of 1:2, the denaturation is completely inhibited. More directly, pelleting experiments have indicated that the binding of phalloidin to actin subunits is saturated at the same molar ratio. The protection of F-actin from heat denaturation or depolymerization with 0.6 M KI is also complete with one molecule of the toxin over two actin subunits. Therefore, it is concluded that the binding ratio of phalloidin and the actin subunit is not 1:1 but 1:2.

Amyloid beta-peptide-induced inhibition of MTT reduction in PC12h and C1300 neuroblastoma cells: effect of nitroprusside.

We have investigated the effect of amyloid beta-peptide (Abeta) in rat pheochromocytoma PC 12h and murine C 1300 neuroblastoma cells by using MTT ¿3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay. Exposure of the cells to Abeta peptides, Abeta1-40 and its fragment Abeta25-35, induced a concentration-dependent inhibition of MTT reduction in both cell lines, and MTT-dependent LDH release due to cell lysis in PC12h cells. We also found that sodium nitroprusside (SNP), a spontaneous nitric oxide (NO) generator, significantly prevented the inhibition of MTT reduction and MTT-dependent LDH release caused by Abeta peptides at 10-100 microM, although a high concentration of SNP (> or = 333 microM) was remarkably toxic by itself. Since the inhibition of MTT reduction caused by Abeta is known as one of the first indicators of its toxicity, these findings suggest that Abeta peptides have a toxic effect in these cell lines, and SNP may attenuate the Abeta peptide-induced toxicity. In regard the mechanisms of the actions of SNP, hydroxylamine which also generates NO and 8-Br-cGMP, a membrane-permeable analogue of cyclic GMP (cGMP), failed to prevent the inhibition of MTT reduction caused by Abeta25-35 in PC12h cells, implying that the effect of SNP may be mediated by the NO-independent pathway. Since potassium ferrocyanide showed a significant prevention at 333 microM although it had toxic effect at this concentration, it is considered that the ferrocyanide portion of the SNP metabolite may be partially involved. The cell death induced by other oxidative insults, such as glutamate and hydrogen peroxide (H2O2), could not be attenuated by SNP in both cell lines. Thus, the observed effect of SNP might not be due to its direct antioxidative action.

beta-Amyloid peptide, substance P, and SEC receptor ligand activate cytoplasmic Ca2+ in neutrophil-like HL-60 cells: effect of chemotactic peptide antagonist BocMLF.

It has been reported that a discrete peptide fragment of beta-amyloid protein, beta A(25-35), and neuropeptide substance P (SP) possessed sequence homology and could bind to the serine protease inhibitor (serpin) enzyme complex (SEC) receptor. Thus, it has been thought that these peptides and SEC receptor ligand might have similar biological activities. In the present study, we found that C-terminal amidated beta A(25-35)-NH2, SP, and the SEC receptor ligand, Phe-Val-Phe-Leu-Met(FVFLM), could induce an increase in the intracellular free Ca2+ concentration ([Ca2+]i) in neutrophil-like human leukemic (HL-60) cells. Pretreatment with pertussis toxin (PTX) potently inhibited the increase in [Ca2+]i stimulated by these peptides, suggesting that these responses might be mediated by PTX-sensitive G-proteins. Furthermore, we examined the effect on these responses of t-butyloxycarbonyl-methionyl-leucyl-phenylalanine (BocMLF), which is a competitive antagonist of chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLF) at its receptor. BocMLF scarcely inhibited the [Ca2+]i increase stimulated by beta A(25-35)-NH2. However, the increase in FVFLM-induced [Ca2+]i was potently inhibited by BocMLF. The results suggest that the [Ca2+]i activation of beta A(25-35)-NH2 may have a different mechanism from that of FVFLM in neutrophil-like HL-60 cells, which is not mediated by the SEC-receptor.

Pharmacological evidence for neurokinin receptors in murine neuroblastoma C1300 cells.

We found that neurokinin A (NKA) and neurokinin B (NKB) induce an increase in the concentration of intracellular free Ca2+ ([Ca2+]i) in murine neuroblastoma C1300 cells (EC50: NKA 87 +/- 13 nM, NKB 97 +/- 15 nM). Substance P (SP) also caused a transient Ca2+ increase, although the potency of SP was much less than that of NKA and NKB. The increase in [Ca2+]i induced by NKA and NKB was inhibited by SR 48,968, a selective antagonist for NK2, and [beta Ala8]NKA(4-10), a selective agonist for NK2, did not stimulate the increase in [Ca2+]i. NKA- and NKB-induced Ca2+ mobilization was not inhibited by CP-96,345 and [Trp7, beta Ala8]NKA(4-10), selective antagonists for NK1 and NK3, respectively. These results suggested that C1300 cells express endogenous NK2 neurokinin receptors that have different features from known NK2 receptors.

Structure-activity studies of heptapeptide derivatives related to substance P, neurokinin A, B and other tachykinins on smooth muscles.

Diverse C-terminal heptapeptide derivatives related to substance P, kassinin, physalaemin, neurokinin A and B were synthesized and the contracting activities on the guinea pig ileum as well as rat duodenum were compared. In the partial sequence of C-terminal of tachykinin peptides, -I-II-Phe-III-Gly-Leu-Met-NH2, the combination of amino acid residues at positions I and III have significant roles in contraction of smooth muscle. For the activation of rat duodenal muscle (SP-E), Asp(I) and aliphatic amino acid(III), and for guinea pig ileal muscle(SP-P), Gln(I) and aromatic amino acid(III) are essential. Moreover, guinea pig ileum is sensitive to a full sequence of neurokinin peptides.

Signaling pathways via NK1 receptors and their desensitization in an AR42J cell line.

Substance P (SP) has been shown to induce phosphatidylinositol (PI) hydrolysis and Ca2+ mobilization in AR42J cells. In this study, we confirmed the expression of NK1 but not NK2 or NK3 receptors in this cell line, and further investigated signaling pathways via NK1 receptors and their desensitization. The activation of NK1 receptors by SP affected neither basal cyclic AMP level nor cyclic AMP accumulation induced by secretin and forskolin, although it stimulated PI hydrolysis. Furthermore, SP induced Ca2+ mobilization even in the absence of extracellular Ca2+, though maximal response was reduced. U73122, a phospholipase C (PLC) inhibitor, nearly abolished Ca2+ response to SP. In addition, SP-induced Ca2+ signaling and PI hydrolysis rapidly desensitized following short exposure to SP, which did not affect the Ca2+ amount in intracellular Ca2+ stores or Ca2+ responses to carbachol and gastrin releasing peptide-10. These findings suggested that NK1 receptors do not couple to adenylate cyclase, although they induce PI response, and that NK1 receptors induce both intracellular Ca2+ release and Ca2+ influx through PLC activation. Ca2+ signaling and PI hydrolysis through NK1 receptors desensitized rapidly after the stimulation, maybe dependent on the modification of NK1 receptors.

Synthesis and biological activity of N-terminal-truncated derivatives of human epidermal growth factor (h-EGF).

To investigate the contribution of the N-terminal sequence of h-EGF to its biological activity and the formation of three intramolecular disulfide bonds by oxidative refolding via air oxidation, five derivatives of h-EGF with a single N-terminal amino acid deletion were synthesized by solid-phase synthesis. The homogeneity of the synthetic peptides was confirmed by analytical reversed-phase HPLC, amino acid analysis, and FAB-MS. The pairing of the three disulfide bridges in synthetic peptides was determined by thermolytic digestion. All N-truncated derivatives of h-FGF formed the correct intramolecular three disulfide linkages during oxidative refolding and had equipotent activity in both EGF receptor binding on A-431 epidermoid carcinoma cells and mitogenesis on NIH-3T3 fibroblast cells, compared with authentic h-EGF. The results suggested that the five residues from N-terminal sequence of h-EGF have no effect on the formation of the correct disulfide linkages in h-EGF and do not exert a significant influence on its biological activity.

Antagonism by GRP(18-27) and substance P analogues on insulin release stimulated by GRP(18-27).

The antagonistic effects of [D-Phe25]gastrin-releasing peptide (GRP)(18-27) and [D-Arg1,D-Pro2,D-Trp7,9,Leu11]substance P (SP) on the stimulation of insulin release by GRP(18-27) from isolated canine pancreas were compared with that of [Ala23]GRP(18-27). The stimulation of insulin release by 1 nM GRP(18-27) was reduced to 24.1% and 15.4% by the prior infusion of 1 microM of [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP and 10 microM of [D-Phe25]GRP(18-27), respectively. Glucagon release by GRP(18-27) was not affected by these peptides using the above concentrations. The results indicate that these peptides are antagonists of bombesin-like peptide receptors on pancreatic B-cells, although the inhibitory activities are lower than that of [Ala23]GRP(18-27).

Effects of taurine and gamma-aminobutyric acid on akinesia and analgesia induced by D-Ala2-Met-enkephalinamide in rats.

Effects of taurine or gamma-aminobutyric acid (GABA) on akinesia and analgesia induced by D-Ala2-Met-enkephalinamide were investigated in rats. Administration of taurine (dose range: 2.375 X 10(-2) M-9.5 X 10(-2)-Met-enkephalinamide were investigated in rats. Administration of taurine (dose range: 2.375 X 10(-2) M-9.5 X 10(-2) M/10 microliters) into the left lateral ventricle 10 min prior to the injection of D-Ala2-Met enkephalinamide (50 microgram/10 microliter) produced a dose-dependent reduction in the duration of akinesia and to some extent of analgesia, as estimated at 30 min and 60 min following the enkephalinamide injection; at the first estimation-time (10 min), taurine did not alter the duration of akinesia or that of analgesia. The median effective dose (ED50) for akinesia determined at 60 min after D-Ala2-Met-enkephalinamide was 5 times greater and that for analgesia assessed at the same time was 1.7 times greater in taurine-treated rats than the respective doses in control animals. Administration of GABA under similar experimental conditions produced a dose-dependent reduction in the duration of analgesia from the initial estimation time (10 min) following the injection of D-Ala2-Met-enkephalinamide. The ED50 for analgesia determined at 30 min after D-Ala2-Met-enkephalinamide was 3 times greater in GABA-treated rats than in control animals. Unlike the effects of taurine, GABA did not alter the duration of akinesia. Neither the duration of akinesia nor that of analgesia was modified by taurine or GABA alone in rats tested 9 min after the injection of each amino acid. These findings suggest that taurine may promote a recovery from both akinesia and analgesia, while GABA decreases only the analgesia induced by D-Ala2-Met-enkephalinamide.