Behaviour studies of the fungicide cymoxanil in two strains of the fungus Botrytis cinerea and in haemolymph of locust and lobster. I. In situ monitoring by internal surface reversed-phase high-performance liquid chromatography.

A method for following the metabolism of the fungicide cymoxanil in various biological media is described. By using a recently developed high-performance liquid chromatographic method, with an internal surface reversed-phase column, it is unnecessary to clean up the sample before analysis. Thus this technique makes monitoring in fungi as well as in arthropod haemolymph easier and faster.

Molecular structure of an apolipoprotein determined at 2.5-A resolution.

The three-dimensional structure of an apolipoprotein isolated from the African migratory locust Locusta migratoria has been determined by X-ray analysis to a resolution of 2.5 A. The overall molecular architecture of this protein consists of five long alpha-helices connected by short loops. As predicted from amino acid sequence analyses, these helices are distinctly amphiphilic with the hydrophobic residues pointing in toward the interior of the protein and the hydrophilic side chains facing outward. The molecule falls into the general category of up-and-down alpha-helical bundles as previously observed, for example, in cytochrome c'. Although the structure shows the presence of five long amphiphilic alpha-helices, the alpha-helical moment and hydrophobicity of the entire molecule fall into the range found for normal globular proteins. Thus, in order for the amphiphilic helices to play a role in the binding of the protein to a lipid surface, there must be a structural reorganization of the protein which exposes the hydrophobic interior to the lipid surface. The three-dimensional motif of this apolipoprotein is compatible with a model in which the molecule binds to the lipid surface via a relatively nonpolar end and then spreads on the surface in such a way as to cause the hydrophobic side chains of the helices to come in contact with the lipid surface, the charged and polar residues to remain in contact with water, and the overall helical motif of the protein to be maintained.

Lom-AG-myotropin: a novel myotropic peptide from the male accessory glands of Locusta migratoria.

A myotropic peptide, termed Lom-AG-myotropin, was isolated from extracts of 4400 accessory gland complexes of males of the locust, Locusta migratoria; the following sequence was derived: Gly-Phe-Lys-Asn-Val-Ala-Leu-Ser-Thr-Ala-Arg-Gly-Phe-NH2. This sequence is completely different from all presently known myotropic peptides from Locusta or other insects. The Lom-AG-myotropin is active on the oviduct and hindgut of Locusta migratoria and Leucophaea maderae. The stimulatory activity is, in both insects, 1000 times greater on the oviduct than on the hindgut, suggesting a specificity for the oviduct.

Isolation, primary structure, and synthesis of locustapyrokinin: a myotropic peptide of Locusta migratoria.

A neuropeptide which stimulates the motility of the cockroach hindgut has been isolated from an extract of 9000 brain-corpora cardiaca-corpora allata-subesophageal 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 primary structure of this myotropic peptide was established as a blocked 16-residue peptide: pGlu-Asp-Ser-Gly-Asp-Gly-Trp-Pro-Gln-Gln-Pro-Phe-Val-Pro-Arg-Leu-NH2. This novel locust peptide was designated as locustapyrokinin, or Lom-PK. Lom-PK was synthesized and shown to have chromatographic and biological properties identical to those of the native material. Lom-PK has a Phe-X-Pro-Arg-Leu-NH2 carboxy terminal in common with leucopyrokinin (or Lem-PK), a blocked myotropic neuropeptide isolated from the cockroach hindgut. The constituent amino acids of this C-terminal are important for biological activity on the Leucophaea hindgut. The primary structure of this novel insect peptide is, however, substantially different from Lem-PK at the amino-terminal sequence.

Sodium channel polypeptides in central nervous systems of various insects identified with site directed antibodies.

Immunoprecipitation, radiophosphorylation and SDS-PAGE autoradiography enable the characterization of sodium channel polypeptides in the central nervous system of insects belonging to four phylogenetically distinct orders: grasshoppers, cockroaches, flies and moth larvae. It has been shown that the insect sodium channels: (1) Are recognized by the previously described (Gordon et al. (1988) Biochemistry 27, 7032-7038) site directed antibodies corresponding to a highly conserved segment linking the homologous domains III and IV in the vertebrate sodium channel alpha subunits. (2) Serve as substrates for phosphorylation by cAMP-dependent protein kinase. (3) Are devoid of disulfide linkage to smaller subunits unlike sodium channels in vertebrate brain. (4) Are glycoproteins as shown in the grasshopper by the decrease of apparent molecular weight following endoglycosidase F treatment and specific binding to the lectins concanavalin A and wheat germ agglutinin. (5) Reveal a diversity with regard to their (a) apparent molecular masses which range from 240 to 280 kDa and (b) V8 proteinase digestion phosphopeptides indicating either differences in the positioning of the enzymatic cleavage and/or phosphorylation sites. These results provide the first evidence for structural diversity of sodium channel subtypes among various insect orders and are compared to their mammalian counterparts.

Isolation, identification and synthesis of locustamyotropin (Lom-MT), a novel biologically active insect peptide.

A peptide that stimulates the spontaneous contractions of the hindgut of Leucophaea maderae has been purified from extracts of brain-corpora cardiaca/corpora allata-subesophageal ganglion complexes of 9000 adult Locusta migratoria and was designated locustamyotropin or Lom-MT. The primary structure of this 12 residue peptide has been determined Gly-Ala-Val-Pro-Ala-Ala-Gln-Phe-Ser-Pro-Arg-Leu-NH2. The C-terminal sequence (Phe-Ser-Pro-Arg-Leu-NH2) is identical to the C-terminal pentapeptide of the pheromone biosynthesis activating neuropeptide, recently isolated from Heliothis zea, and is also similar to the C-terminal of leucopyrokinin of Leucophaea. Synthetic Lom-MT showed biological as well as chemical characteristics, indistinguishable from those of native Lom-MT. In locust preparations, Lom-MT provoked an increase in frequency, amplitude and tonus of contractions of the oviduct, but was inactive in the same conditions on the locust hindgut preparation.

Locustatachykinin I and II, two novel insect neuropeptides with homology to peptides of the vertebrate tachykinin family.

Two myotropic peptides termed locustatachykinin I (Gly-Pro-Ser-Gly-Phe-Tyr-Gly-Val-Arg-NH2) and locustatachykinin II (Ala-Pro-Leu-Ser-Gly-Phe-Tyr-Gly-Val-Arg-NH2) were isolated from brain-corpora cardiaca-corpora allata-suboesophageal ganglion extracts of the locust, Locusta migratoria. Both peptides exhibit sequence homologies with the vertebrate tachykinins. Sequence homology is greater with the fish and amphibian tachykinins (up to 45%) than with the mammalian tachykinins. In addition, the intestinal myotropic activity of the locustatachykinins is analogous to that of vertebrate tachykinins. The peptides discovered in this study may just be the first in a whole series of substances from arthropod species to be identified as tachykinin family peptides. Moreover, both chemical and biological similarities of vertebrate and insect tachykinins substantiate the evidence for a long evolutionary history of the tachykinin peptide family.

Isolation and structure elucidation of a novel 5-kDa peptide from neurohaemal lobes of the corpora cardiaca of Locusta migratoria (Insecta, Orthoptera).

Two predominant peptides have been isolated from neurohaemal lobes of corpora cardiaca of 8000 adults of Locusta migratoria. Both peptides have been unambiguously characterized by automated peptide microsequencing and liquid secondary-ion mass spectrometry as a 50-residue peptide (5K peptide) and a 48-residue isologue (5K' peptide). Computer search of sequence data banks did not reveal any significant similarity with other identified proteins. The 5K peptides are remarkably rich in alanine residues (25%) and contain a stretch of five consecutive alanines. This structure suggests that these molecules could correspond to spacer peptides. This assumption is corroborated in the accompanying paper [Lagueux et al. (1990) Eur. J. Biochem. 187, 249-254] on the molecular cloning of the precursor protein which attributes to the 5K peptides a role analogous to that of the C peptides of insulins.

The structurally similar neuropeptides adipokinetic hormone I and II are derived from similar, very small mRNAs.

The grasshopper neuropeptides adipokinetic hormone (AKH) I and II were among the first of an extensive family of structurally similar arthropod hormones and neuroregulators to be isolated and sequenced. This paper reports the cloning of cDNAs derived from the unusually small mRNAs (550 bases) which code for the precursors of AKH I and II from Schistocerca nitans. Sequence analysis of the cDNAs indicates that AKH I and II are derived from small precursor proteins (63 and 61 amino acids) which are 55% identical in amino acid sequence. Each contains a 22-amino acid hydrophobic leader sequence followed by the AKH I or II sequence and an additional 28-amino acid carboxyl-terminal peptide of unknown function. Significant homology at the nucleic acid level (64% identity) is confined to the coding region of the mRNA sequences. Preliminary DNA blot analyses suggest that a single gene codes for each, and that the genes for AKH I and II may be linked. Genomic blots from various tissues fail to suggest that the high level of expression of AKH in the corpora cardiaca is due to tissue specific gene amplification.

Functional renaturation of receptor polypeptides eluted from SDS polyacrylamide gels.

In order to gain further support for the concept that a homo-oligomeric protein-complex may be sufficient to form a functional ligand-activated ion channel and to explore additional possibilities for the reconstitution of channel activity, a single polypeptide band of the purified neuronal AChR from insects has been electroeluted from SDS-polyacrylamide gels, the SDS removed and the polypeptides incorporated into liposomes. Liposomes were fused into planar lipid bilayers which were subsequently analysed for channel activity. Fluctuations of cation-channels were detected after addition of agonists (carbamylcholine); channel activity was blocked by antagonists (d-tubocurarine). The channels formed by electroeluted polypeptides gave conductance values, as well as kinetic data, quite similar to channels formed by the native receptor protein. Sedimentation experiments using sucrose density gradient centrifugation revealed that a considerable portion of the electroeluted polypeptides assembled during the reconstitution process to form oligomeric complexes with a sedimentation coefficient of about 10 S; thus resembling the native receptor complex.

Plasma-desorption mass spectrometry as an aid in protein sequence determination. Application of the method on a cuticular protein from the migratory locust (Locusta migratoria).

The complete amino acid sequence of a structural protein, protein 8, isolated from the pharate cuticle of the locust Locusta migratoria was determined. Protein 8 contains 148 amino acid residues and has an Mr of 15,224. By the extensive use of information obtained by plasma-desorption mass spectrometry (p.d.m.s.) it was possible to reduce the need for conventional sequence determination and to improve the reliability of the results. On the basis of the determined Mr of the intact protein all the peptides that constitute the complete sequence could be isolated from a time-course enzymic digestion. The isolated peptides were sequenced by using a combination of Edman degradation and carboxypeptidase digestion monitored by p.d.m.s. The alignment of the peptides was established from the time-course digestion and further verified by a second enzymic digestion. The primary structure of the protein consists of two hydrophilic and two hydrophobic regions. The hydrophobic regions are enriched in alanine, valine and proline and dominated by a repetitive sequence Ala-Ala-Pro-(Ala/Val). The sequence strengthens the view that the cuticle proteins belong to a unique family of structural proteins.

Isolation and structure of two novel 6-kDa dimeric peptides from the corpora cardiaca of the insect Locusta migratoria. Molecular mass determination by mass spectrometry.

We have isolated two major 6-kDa peptides from extracts of corpora cardiaca of adult females of Locusta migratoria. These peptides have been characterized by peptide sequencing and liquid secondary-ion mass spectrometry. They are structurally related dimers, one (6278.5 Da) being a homodimer (A-A chains), the other (6280.5 Da) being a heterodimer (A-B chains). A 60% similarity exists between the A and B chains. Both peptides have been chemically synthesized and the synthetic compounds appeared to be identical to the native ones. Polyclonal antibodies raised against each of these peptides demonstrated that they were contained within the secretory granules of the intrinsic cells of the glandular lobes of the corpora cardiaca. The physiological significance of these two peptides is unknown but, using the synthetic peptides, we are currently probing their biological role.

Isolation and reconstitution of the high-affinity choline carrier.

Monoclonal antibodies, which block the high-affinity uptake of choline in synaptosomal ghosts, have been used to purify a membrane polypeptide (80 kDa) from insect synaptosomal membranes. This isolated protein was found to catalyse the sodium-dependent, hemicholinium-sensitive accumulation of choline after reconstitution into liposomes, thus, apparently represents the high-affinity choline transporter.

Amino acid sequence of locust neuroparsins.

Neuroparsins A and B were isolated from the nervous part of the corpus cardiaca of Locusta migratoria via a two-step purification procedure. Both consist of two polypeptide chains linked by disulfide bridges. The N-terminal sequence of both native neuroparsins was determined: the N-terminal end of neuroparsin B was unique while that of neuroparsin A showed three different sequences. These sequences were that of neuroparsin B and two others having five and two extra N-terminal residues. Neuroparsin B was found as a homodimer and the complete sequence of the monomer, determined from peptide fragments generated by treatment with cyanogen bromide and endoprotease Glu-C, comprises 78 residues.

Further characterisation of the [35S]-TBPS binding site of the GABA receptor complex in locust (Schistocerca gregaria) ganglia membranes.

1. Using homogenates of supraoesophageal ganglia from locust we observed specific binding of [35S]-TBPS which was linear with protein concentration up to 7 mg/ml, showed a pH optimum at pH 9.0 and was linear with NaCl concentration up to 350 mM. 2. Kinetic analysis of the binding showed positive cooperativity as a result of changes in on and off-rates with occupation of the binding site by the ligand. The apparent KD = 417 nM and Bmax = 1083 fmol/mg of membrane protein were calculated using a computer program for dose-response curve fitting. 3. The binding was enhanced by GABA, pentobarbital and benzodiazepines. Picrotoxinin had no effect on the binding at 0.1 mM. Only the cage convulsants TBPS and IBP were able to displace the binding. 4. Whilst the characteristics of the binding are similar to those reported for house fly thorax and abdomen preparations they are significantly different from those reported for house fly head, cockroach nerve cord and rat brain.

The presence of a pregnenolone-binding factor in the copulatory organ of the migratory locust, Locusta migratoria migratorioides R. & F.

The presence of binding sites for nonecdysteroid steroids was investigated in the cytosol of several tissues of the migratory locust, Locusta migratoria migratorioides. Binding of androgens was not observed. Most tissues, however, showed nonsaturable binding of estrogens and in some tissues saturable progestin binding could be demonstrated. A pregnenolone binder, that was found to be present in the male copulatory organ, was further studied. It showed a dissociation constant of 4.4 (+/- 1.6) X 10(-8) M. This is the first report of a nonecdysteroid steroid-binding factor in an insect tissue.

Immunohistological demonstration of a substance related to neuropeptide Y and FMRFamide in the cephalic and thoracic nervous systems of the locust Locusta migratoria.

A neuropeptide related to the mammalian neuropeptide Y (NPY) is present in various neurosecretory cells (NSC) of the cephalic and thoracic nervous systems of the insect Locusta migratoria. Immunoreactive perikarya are detected in the protocerebrum, tritocerebrum, optic lobes and the suboesophageal and thoracic ganglia. They give rise to many immunoreactive processes that ramify extensively throughout the neuropiles. In the brain, prominent axon bundles tightly surround the tractus I to the corpora cardiaca. This fiber pattern suggests that the NPY-like substance may have a neuromodulator and/or neurotransmitter function. This substance may also have a neurohormonal role, since some immunoreactive tracts penetrate into neurohaemal organs via the nervi corporis cardiaci II and the thoracic median nerves. NCS containing NPY-like neuropeptide also display an FMRFamide-like immunoreactivity (except for the abdominal part of the metathoracic ganglion). NPY or FMRFamide antisera are not inactivated after preabsorption with FMRFamide or NPY, respectively. It might therefore be inferred that in locust NSC these two antisera recognize two distinct antigenic sites belonging either to a large polypeptide, or to two distinct neuropeptides.

NPY-like peptides occur in the nervous system and midgut of the migratory locust, Locusta migratoria and in the brain of the grey fleshfly, Sarcophaga bullata.

The distribution of the NPY-like substances in the nervous system and the midgut of the migratory locust, Locusta migratoria and in the brain of the grey fleshfly, Sarcophaga bullata was determined by immunocytochemistry using an antiserum directed against synthetic porcine NPY. The peroxidase-antiperoxidase procedure revealed that NPY immunoreactive cell bodies and nerve fibers were observed in the brain, optic lobes, corpora cardiaca, suboesophageal ganglion and ventral nerve cord of the locust and in the brain, optic lobes and suboesophageal ganglion of the fleshfly. In the locust midgut, numerous endocrine cells and nerve fibers penetrating the outer musculature contained NPY-like immunoreactivity. The concentrations of NPY immunoreactive material in acetic acid extracts of locust brain, optic lobes, thoracic ganglia, ovaries and midguts was measured using a specific radioimmunoassay technique. The dilution curves of the crude tissue extracts were parallel to the standard curve. The highest amount of NPY-like immunoreactivity was found in the locust ovary and midgut. Reverse-phase high-performance liquid chromatography (RP-HPLC) and radioimmunoassay were used to characterize the NPY-like substances in the locust brain and midgut. HPLC-analysis revealed that NPY-immunoreactivity in the locust brain eluted as three separate peaks. The major peak corresponded to a peptide less hydrophobic than synthetic porcine NPY. RP-HPLC analysis of midgut extracts revealed the presence of an additional NPY-immunoreactive peak which had a retention time similar to the porcine NPY standard. The present data show the existence of a widespread network of NPY immunoreactive neurons in the nervous system of the locust and the fleshfly. Characterization of the immunoreactive substances indicates that peptides similar but not identical to porcine NPY are present in the central nervous system and midgut of insects.

FMRFamide-like immunoreactivity in the metathoracic ganglion of the locust (Schistocerca gregaria).

The distribution of FMRFamide-like immunoreactivity in the metathoracic ganglion of the locust, Schistocerca gregaria, has been investigated in serial semithin transverse sections with the use of the peroxidase-antiperoxidase (PAP) technique. The topographical distribution of approximately 120 immunopositive neurons was established. Antiserum against bovine pancreatic polypeptide (BPP) stains the same ganglionic cells as FMRFamide-antiserum, yet this staining is largely blocked after preabsorption to FMRFamide. A comparison of these results with those from other studies suggests that there may be more than one type of endogenous RFamide-like peptide.

Sequence analyses of two neuropeptides of the AKH/RPCH-family from the lubber grasshopper, Romalea microptera.

Two neuropeptides with adipokinetic activity in Locusta migratoria and hypertrehalosaemic activity in Periplaneta americana were purified by high-performance liquid chromatography from the corpus cardiacum of the lubber grasshopper, Romalea microptera. The sequences of both peptides, designated Ro I and Ro II, were determined by gas-phase sequencing employing Edman degradation after the N-terminal pyroglutamate residue was enzymatically deblocked, as well as by fast atom bombardment mass spectrometry. Ro I was found to be a decapeptide with the primary structure: pGlu-Val-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2, whereas Ro II is an octapeptide with the structure: pGlu-Val-Asn-Phe-Ser-Thr-Gly-Trp-NH2. Ro II is identical with AKH-G isolated from the cricket Gryllus bimaculatus. Synthetic materials having the assigned structures were found to be chromatographically, mass spectrometrically, and biologically indistinguishable from the natural peptides, confirming the sequences and establishing the Romalea peptides as members of the AKH/RPCH-family of peptides.