Isolation, identification and synthesis of locustamyoinhibiting peptide (LOM-MIP), a novel biologically active neuropeptide from Locusta migratoria.

A novel peptide termed locustamyoinhibiting peptide (LOM-MIP) was isolated from brain-corpora cardiaca-corpora allata-suboesophageal ganglion extracts of the locust, Locusta migratoria. The primary structure of this nonapeptide has been determined Ala-Trp-Gln-Asp-Leu-Asn-Ala-Gly-Trp-NH2. LOM-MIP suppresses the spontaneous contractions of the hindgut and oviduct of Locusta migratoria and of the hindgut of Leucophaea maderae. This novel peptide is, however, structurally different from leucomyosuppressin, a hindgut suppressing peptide isolated from Leucophaea maderae heads. LOM-MIP has a Gly-TrpNH2 carboxy-terminal in common with APGWamide, a penis retractor muscle inhibiting peptide isolated from the snail, Lymnea stagnalis. In addition, it shows carboxy-terminal sequence similarities with locust AKH II which ends in AGWamide. No sequence similarities were found with other vertebrate or invertebrate peptides. Synthetic LOM-MIP showed biological as well as chemical characteristics indistinguishable from those of native LOM-MIP.     

Isolation,identification and synthesis of locustamyotropin II,an additional neuropeptide of Locusta migratoria: Member of the cephalomyotropic peptide family

An eight residue neuropeptide (Glu-Gly-Asp-Phe-Thr-Pro-Arg-Leu-NH₂) has been isolated from an extract of 9000 brain corpora cardiaca-corpora allata-suboesophageal ganglion complexes of Locusta migratoria. Biological activity was monitored during HPLC purification by observing the myotropic effect of column fractions on the isolated hindgut of Leucophaea maderae. The peptide designated as locustamyotropin II, or Lom-MT II according to Raina and Gäde (Insect Biochem.18, 785–787, 1988), has a Phe-X-Pro-Arg-Leu-NH₂ carboxyl-terminal in common with the previously identified locustamyotropin I. Locustamyotropin II is also related to leucopyrokinin (Lem-PK), a blocked myotropic neuropeptide isolated from cockroach heads. Both peptides have identical carboxyterminal pentapeptide sequences. The constituent amino acids of this C-terminal are important for biological activity on the Leucophaea hindgut. Lom-MT II differs from Lem-PK in the first three aminoterminal residues. In contrast to Lem-PK and like Lom-MT I, the novel locust peptide is not N-terminally blocked. Lom-MT II has a stimulatory effect on the motility of the oviduct of Locusta but not on the hindgut.     

Neurotensin, a biologically active peptide.

Neurotensin, a tridecapeptide appears to be localized in various parts of the brain and gut. A high affinity binding component of neurotensin to brain membranes, synaptosomes and mast cells has been reported. After peripheral administration the peptide exerts a medley of effects which appear directed mainly to glucose metabolism. In addition, complex vascular effects have also been noted including hypotensin, cyanosis, vasodilation and increased permeability. The peptide may also be associated with inflammatory events. Complex effects upon the secretion of anterior pituitary tropic hormones have been observed.After central administration neurotensin exerts several effects all of which appear to be sufficiently explained by the potent hypothermic action. The resolution of the question of which, if any, of the actions of neurotensin are involved in physiological regulation has not been achieved.     

A novel 31-amino-acid-length endothelin, ET-1(1-31), can act as a biologically active peptide for vascular smooth muscle cells

Human chymase produces a novel endothelin-1 with 31 amino-acid length ?ET-1(1-31)?, which is longer than conventional ET-1, ?ET-1(1-21)?. The aim of our study was to investigate the role of ET-1(1-31) on porcine coronary vascular smooth muscle cell (VSMC). Although the increase in [Ca(2+)](i) by ET-1(1-31) was 10 times weaker than that of ET-1(1-21), ET-1(1-31) showed equivalent potency in VSMC proliferation, c-fos/c-myc mRNA expression and cell cycle analysis with ET-1(1-21). ET-1(1-31) significantly induced expression of cyclin D1 but not those of cyclin D2 or D3. These effects were specifically inhibited by BQ485, an ET(A) receptor antagonist, although that of ET-1(1-21) was not specific to BQ485, suggesting different receptor specificity from ET-1(1-21). These results indicate that ET-1(1-31) also can involve a VSMC proliferation process such as atherosclerosis, although it has weaker vasoconstricting potency and different receptor subtypes on VSMC from those of ET-1(1-21).     

Isolation,identification, and synthesis of Mas-MG-MT I,a novel peptide from the larval midgut of Manduca sexta (lepidoptera: Sphingidae)

A five-residue myotropic peptide, Manduca sexta midgut myotropin I (Mas-MG-MT I), was isolated from an extract of 800 midguts of fifth instar larvae of the tobacco hornworm, Manduca sexta. It was purified by reverse phase and normal phase HPLC. Myotropic activity was screened by a heterologous Locusta migratoria oviduct bioassay. Sequence analysis, amino acid composition analysis, and comparison of candidate synthetic peptides in the amide and acid form revealed the following primary structure: Ala-Glu-Pro-Tyr-Thr-NH₂. This is the first fully identified peptide isolated directly from the midgut of an insect species. Few significant sequence homologies with known vertebrate and invertebrate peptides have been found. © 1995 Wiley-Liss, Inc.     

Isolation of Leu-Pro-Pro-Gly-Pro-Leu-Pro-Arg-Pro-NH2 (Antho-RPamide), an N-terminally protected, biologically active neuropeptide from sea anemones.

Using a radioimmunoassay against the C-terminal sequence Arg-Pro-NH2 (RPamide), we have isolated the peptide Leu-Pro-Pro-Gly-Pro-Leu-Pro-Arg-Pro-NH2 (Antho-RPamide) from an extract of the sea anemone Anthopleura elegantissima. Antho-RPamide is located in neurons of sea anemones. Application of low concentrations of Antho-RPamide to tentacle preparations of sea anemones strongly increased the frequency and duration of spontaneous contractions, suggesting that this peptide is involved in neurotransmission. Antho-RPamide has a free N-terminus, yet its X-Pro-Pro sequence makes it relatively resistant to degradation by nonspecific aminopeptidases. Thus, we have discovered another strategy by which sea anemones protect the N-termini of their bioactive neuropeptides.     

Isolation, identification and synthesis of novel oviductal motility stimulating head peptide in the Colorado potato beetle, Leptinotarsa decemlineata

A novel myotropic peptide was isolated from an extract of 10,000 heads of adult Leptinotarsa decemlineata by means of high performance liquid chromatography (HPLC). The peptide stimulates the contractions of the oviduct of Leptinotarsa as well as that of Locusta migratoria. Gas phase sequencing and comparison of candidate synthetic peptides in the amide and acid form revealed the following primary structure: Ile-Ala-Tyr-Lys-Pro-Glu-NH2. This new peptide has a molecular weight of 720 Da and has been named Led OVM. Led OVM does not exhibit significant sequence homology with any known vertebrate or invertebrate peptide. Sixteen additional myotropic factors were also separated by means of HPLC, but were as yet not recovered in amounts large enough for them to be sequenced.     

Isolation, gene expression and solution structure of a novel moricin analogue, antibacterial peptide from a lepidopteran insect, Spodoptera litura

An antibacterial peptide was isolated from a lepidopteran insect, Spodoptera litura. The molecular mass of this peptide was determined to be 4489.55 by matrix assisted laser desorption/ionization-time of flight mass (MALDI-TOF MS) spectrometry. The peptide consists of 42 amino acids and the sequence has 69-98% identity to those of moricin-related peptides, antibacterial peptides from lepidopetran insects. Thus, the peptide was designated S. litura (Sl) moricin. Sl moricin showed a broad antibacterial spectrum against Gram-positive and negative bacteria. Sl moricin gene was inducible by bacterial injection and expressed tissue-specifically in the fat body and hemocytes. Furthermore, the solution structure of Sl moricin was determined by two-dimensional (2D) 1H-nuclear magnetic resonance (NMR) spectroscopy and hybrid distance geometry-simulated annealing calculation. The tertiary structure revealed a long alpha-helix containing eight turns along nearly the full length of the peptide like that of moricin, confirming that Sl moricin is a new moricin-like antibacterial peptide. These results suggest that moricin is present not only in B. mori but also in other lepidopteran insects forming a gene family.     

Isolation, amino acid sequence, and synthesis of dermaseptin, a novel antimicrobial peptide of amphibian skin

A 34-residue antimicrobial peptide named dermaseptin was purified to homogeneity from amphibian skin by a 3-step protocol involving molecular sieve filtration, ion-exchange chromatography, and reversed-phase high-performance liquid chromatography. The complete amino acid sequence of dermaseptin, ALWKTMLKKLGTMALHAGKAALGAAADTISQGTQ, was determined by automated Edman degradation of the peptide and of fragments generated by trypsin. Fast atom bombardment mass spectra of dermaseptin gave a protonated molecular ion m/z 3455.4 which matched the theoretical molecular weight predicted from the amino acid sequence. Dermaseptin was synthesized by the solid-phase method. The synthetic replicate was shown to be indistinguishable from natural dermaseptin with respect to chromatographic properties, amino acid sequence determination, and mass spectrometry analysis. Dermaseptin is a water-soluble, thermostable, and nonhemolytic peptide endowed with highly potent antimicrobial activity against pathogenic fungi at micromolar concentration. Circular dichroism spectra of dermaseptin in hydrophobic media indicated 80% alpha-helical conformation, and predictions of secondary structure suggested that dermaseptin can be configured as an amphiphatic alpha-helix spanning over residues 1-27, a structure that perturbs membrane functions regulating water flux.     

Isolation and identification of two new biologically active norditerpene dilactones from Aspergillus wentii

Two new biologically-active norditerpenoid dilactones were purified from culture extracts of Aspergillus wentii and assigned the trivial names wentilactone A and wentilactone B. The absolute chemical structure of wentilactone A was determined by single crystal X-ray diffraction and circular dichroism. The structure of wentilactone B was determined by ¹H and ¹³C NMR analyses. Wentilactone A had an ld₅₀ of 7.0 mg/kg when administered orally to 1-day-old chickens. Both metabolites inhibited growth in wheat coleoptile bioassays.     

Structure of a biologically active neurotensin-related peptide obtained from pepsin-treated albumin(s)

Using a radioimmunoassay toward the COOH-terminal region of neurotensin, an immunoreactive and biologically active neurotensin-related peptide (NRP) has been isolated from pepsin-treated fractions of bovine, canine, human, and rat plasma. Bovine NRP was identified as H-Ile-Ala-Arg-Arg-His-Pro-Tyr-Phe-Leu-OH, which is similar in structure to both neurotensin and angiotensin I. Canine and human NRP also had the above amino acid composition, whereas that obtained from rat plasma had valine substituted for isoleucine. At their concentrations in pepsin-treated plasmas (2-6 microM) rat, human and canine NRP were shown to increase vascular permeability when injected intradermally into rats and to release histamine from rat mast cells in vitro. The pure peptides also cross-reacted very effectively at nanomolar concentrations in a radioreceptor assay for neurotensin. The protein(s) which liberated NRP upon pepsin treatment were purified about 7-fold and shown to behave like albumin during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and high pressure liquid chromatography on muBondapak C4. In addition, the purified preparations were found to react with anti-albumin antisera during immunodiffusion. Although the amino acid sequence of NRP was not found in albumin, a partial sequence homology was noted for NRP and various segments of bovine albumin. Using V8 protease, glutamyl residues were shown to lie within 3-4 amino acids of each end of NRP, as also occurs for the related segments in albumin. These results suggest that a subset of albumin-related protein(s) could serve as precursor(s) to biologically active neurotensin-related peptide(s).     

Isolation,identification and localization of a second beetle antidiuretic peptide

We isolated from head extracts of Tenebrio molitor a peptide that inhibits fluid secretion by the Malpighian tubules of this insect. This second antidiuretic factor, ADFb, like the previously published ADFa, works through cyclic GMP as a second messenger. It has primary structure Tyr-Asp-Asp-Gly-Ser-Tyr-Lys-Pro-His-Ile-Tyr-Gly-Phe-OH with an EC(50) of approximately 240 pM in a fluid secretion assay. This peptide is now the second sequenced endogenous insect ADF which inhibits Malpighian tubule fluid secretion. Immunohistochemical techniques show that the peptide is localized in the brain; it appears to be produced mainly in two pairs of bilaterally symmetrical cells in the protocerebrum.     

Isolation and identification of pelteobagrin, a novel antimicrobial peptide from the skin mucus of yellow catfish (Pelteobagrus fulvidraco)

Fish skin mucus has recently been recognized to be a potential source of antimicrobial peptides, which provides the first line of defense against invading pathogens. This study reports the purification and characterization of a novel linear antimicrobial peptide, pelteobagrin, from the skin mucus of yellow catfish (Pelteobagrus fulvidraco, Richardson). Pelteobagrin is 20 amino acids in length (GKLNLFLSRLEILKLFVGAL) and shows no clear homology with any known bioactive peptides. MALDI-TOF MS indicated the molecular mass of the purified peptide was 2244.4 Da, which is in good agreement with pelteobagrin's predicted molecular weight of 2244.8 Da. Pelteobagrin exhibited antibacterial activity against Gram-positive and Gram-negative bacteria as well as fungi, and the activity was relatively salt-insensitive as it was not affected by NaCl concentrations of up to 137 mM. Moreover, pelteobagrin displayed no hemolytic activity to rabbit red blood cells. Transmission electron microscopy suggested that pelteobagrin might kill bacteria via acting on both the cell wall and the cytoplasmic membrane of bacteria. These results suggest that pelteobagrin might be involved in the innate defense system in yellow catfish.     

Comparison of methods for thermolysin-catalyzed peptide synthesis including a novel more active catalyst

This is a comparative study of the performance of thermolysin for enzymatic peptide synthesis by reversed hydrolysis in several different reaction systems. Z-Gln-Leu-NH(2) was synthesized in acetonitrile containing 5% water (with various catalyst preparation methods) as well as by the "solid-to-solid" and frozen aqueous methods. Reaction rates (values in nanomoles per minute per milligram) in acetonitrile depended significantly on the method of addition of enzyme: (a) direct suspension in the reaction mixture as freeze-dried powders gave 60 to 95; (b) addition as an aqueous solution, so that enzyme precipitates on mixing with acetonitrile, gave 230; (c) addition as an aqueous suspension gave a remarkable increase in reaction rates (up to 780); (d) immobilized enzymes (adsorbed at saturating loading on celite, silica, Amberlite XAD-7, or polypropylene, then dried by propanol rinsing) all gave <230. It is postulated that, starting with the enzyme already in the form of solid particles in aqueous buffer, there is a minimum chance of alteration of its optimal conformation during transfer to the organic medium. For solid-to-solid synthesis with 10% water content we found initial rates of 670 under optimized conditions. In frozen aqueous synthesis, rates were <10. Equilibrium yields were always around 60% in low water organic solvent, whereas they were found to >80% in the aqueous systems studied.     

Folding of the nascent peptide chain into a biologically active protein

The refolding of denatured proteins with complete sequences may not be fast enough to account for the in vivo folding of growing peptide chains during biosynthesis. As some peptide fragments have secondary structures not unlike those of the corresponding segments in the intact molecules and native disulfide bonds of some proteins can form cotranslationally, it is suggested that the folding of the nascent chain begins early during synthesis. However, further adjustments may be necessary during chain elongation and after posttranslational modifications of the completed peptide chain to generate the native conformation of a biologically active protein.     

Insect immunity. Isolation from a coleopteran insect of a novel inducible antibacterial peptide and of new members of the insect defensin family.

Injection of heat-killed bacteria into larvae of the large tenebrionid beetle Zophobas atratus (Insecta, Endopterygota, Coleoptera) results in the appearance in the hemolymph of a potent antibacterial activity as evidenced by a plate growth inhibition assay. We have isolated three peptides (A-C) from this immune hemolymph which probably account for most of this activity. Their primary structures were established by a combination of peptide sequencing and molecular mass determination by mass spectrometry. Peptide A, which is bactericidal against Gram-negative cells, is a 74-residue glycine-rich molecule with no sequence homology to known peptides. We propose the name coleoptericin for this novel inducible antibacterial peptide. Peptides B and C are isoforms of a 43-residue peptide which contains 6 cysteines and shows significant sequence homology to insect defensins, initially reported from dipteran insects. This peptide is active against Gram-positive bacteria. The results are discussed in connection with recent studies on inducible antibacterial peptides present in the three other major orders of the endopterygote clade of insects: the Lepidoptera, Diptera, and Hymenoptera.     

Isolation of a biologically active low-molecular-mass chromium compound from rabbit liver

A low-molecular-mass chromium-binding substance (LMCr), which is recognized as a detoxification ligand of chromium, was isolated from the livers of rabbits injected intravenously with K2Cr2O7 (200 mumol Cr/kg body wt) as a biologically active form. LMCr appears as an anionic, organic Cr compound with a relative molecular mass of 1500. It is composed of glutamic acid or glutamine, glycine, cysteine and aspartic acid or asparagine with a Cr/amino-terminal residue ratio of 4:1. The purified LMCr (10-300 ng Cr/ml) shows in vitro activities comparable to those of glucose tolerance factor in relation to insulin action. In the presence of insulin it enhances [U-14C]glucose conversion to 14CO (23-30% up) in rat epididymal adipocytes above the value obtained with insulin alone. LMCr also stimulates the rate of [3-3H]glucose incorporation into lipid by 30-40% with insulin or by 15-23% without insulin, as compared with the basic value obtained with insulin alone or without insulin. These findings suggest that LMCr plays essential roles in both glucose metabolism and detoxification of invaded Cr in the body.