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.     

Sugar binding properties of the vitellogenic proteins in the Colorado beetle,demonstrated by hemagglutination and precipitation experiments

Summary The sugar binding properties of 2 important vitellogenic proteins in Colorado beetle hemolymph were demonstrated by hemagglutination and precipitation experiments. The agglutination of human red blood cells by the hemolymph of reproducing females was observed up to a hemolymph dilution of 1/256, irrespective of the blood-group. It increased significantly after trypsinization of the crythrocytes. Vitellogenin 1 was identified as the hemagglutinin. Hemagglutination and hemagglutination inhibition tests showed that this protein has a low affinity for hexosamines and a higher affinity for sulfated polysaccharides. Precipitation tests demonstrated that besides vitellogenin, another major yolk protein, chromoprotein 2, reacts with sulfated polysaccharides. The possibility that there is a specific reaction of the vitellogenic proteins with well defined saccharides on the oocyte surface is discussed. This lectintype reaction may explain the selectivity of yolk precursor endocytosis.     

The amphipathic helix in the exchangeable apolipoproteins: a review of secondary structure and function.

Site-directed mutagenesis and other molecular biology-based techniques are now available for probing the amphipathic alpha helix structural motif in the exchangeable apolipoproteins. Here we survey the published literature on lipid-binding and functional domains in apolipoproteins A-I, A-II, A-IV, C-I, C-II, C-III, and E and compare these results with recently developed computer methods for analysis of the location and properties of amphipathic helixes. This comparison suggests that there are at least three distinct classes of amphipathic helixes (classes A, Y, and G*) in the exchangeable apolipoproteins whose distribution varies within and between the seven apolipoproteins. This comparison further suggests that lipid affinity resides largely in class A amphipathic helixes (Segrest, J. P., et al. 1990. Proteins. 8: 103) and that variations in structure and/or numbers of class A domains in individual apolipoproteins allow a range of lipid affinities from high to low. The positions of the four alpha helixes recently shown to form a 4-helix bundle globular structure in apoE (Wilson, C., et al. 1991. Science. 252: 1817) correspond closely to the four amino-terminal class G* amphipathic helixes of apoE identified by our computer analysis. It is of particular interest, therefore, that all of the exchangeable apolipoproteins except apoA-II and C-I, contain amphipathic helixes of class G*. Additional implications of amphipathic helix heterogeneity for the structure and function of the exchangeable apolipoproteins will be discussed.     

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.     

A novel method for the quantitative extraction of juvenile hormones from insects including the silverfish, Thermobia domestica

An improved procedure for extraction of juvenile hormones (JH) from large quantities of insects is described. It consists of the following steps. The lyophilised insects are homogenized in cold methanol and, after storage overnight, the debris is removed by filtration and the methanol is evaporated. For the next two steps the residue is dissolved in ether and by adding either methanol in the first step, or acetone in the second step, a precipitate is formed which is removed by filtration. With this method, 2 to 7 times less lipids are extracted than with usual methods involving ether or ethyl acetate, but more than 99.5 per cent of the JH is extracted at least in H. cecropia. The JH activity of the extracts is at least 10 times higher than extracts prepared with ether or ethyl acetate alone.     

Mass spectrometric evidence for the deficiency in the dark-color-inducing hormone,.

A factor present in the brain and corpus cardiacum responsible for the induction of dark colour in Locusta migratoria was recently isolated and identified from the corpora cardiaca of normally pigmented locusts. The purification of this factor, designated as [His7]-corazonin was monitored using an albino mutant from a laboratory colony of an Okinawa (Japan) strain. In this study, we provide unequivocal mass spectrometric evidence that the brain and the corpora cardiaca of this albino Locusta mutant are deficient in [His7]-corazonin. Previously, [His7]-corazonin was shown to be responsible for the induction of dark body colour patterns as observed in crowded locusts. Using nanoflow-liquid chromatography-mass spectrometry, we demonstrated that this dark colour-inducing hormone is, however, present in the corpora cardiaca of solitary locusts (Schistocerca gregaria). Arch.     

Conformational analysis of lipid-associating proteins in a lipid environment

Two major types of helical structures have been identified in lipid-associating proteins, being either amphipathic or transmembrane domains. A conformational analysis was carried out to characterize some of the properties of these helices. These calculations were performed both on isolated helices and in a lipid environment. According to the results of this analysis, the orientation of the line joining the hydrophobic and hydrophilic centers of the helix seems to determine the orientation of the helix at the lipid/water interface. The calculation of this parameter should be useful to discriminate between an amphipathic helix, parallel to the interface and a transmembrane helix orientated perpendicularly. The membrane-spanning helices are completely immersed in the phospholipid bilayer and their length corresponds to about the thickness of the hydrophobic core of the DPPC bilayer. The energy of interaction, expressed per phospholipid is significantly higher for the transmembrane compared to the amphipathic helices. For the membrane-spanning helices the mean energy of interaction is higher than the interaction energy between two phospholipids, while it is lower for most amphipathic helices. This might account for the stability of these protein-anchoring domains. This computer modeling approach should usefully complement the statistical analysis carried out on these helices, based on their hydrophobicity and hydrophobic moment. It represents a more refined analysis of the domains identified by the prediction techniques and stress the functional character of lipid-associating domains in membrane proteins as well as in soluble plasma lipoproteins.     

Purification and characterization of 1-SST, the key enzyme initiating fructan biosynthesis in young chicory roots (Cichorium intybus)

A genuine 1-SST (sucrose:sucrose 1-fructosy] transferase, EC 2.4.1.99) was purified and characterized from young chicory roots ( Cichorium intybus L. var. foliosum cv. Flash) by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, anion and cation exchange chromatography. This protocol produced a 63-fold purification and a specific activity of 4.75 U (mg protein)⁻¹. The mass of the enzyme was 69 kDa as estimated by gel filtration. On SDS-PAGE apparent molecular masses of 49 kDa (α-subunit) and 24 kDa (β-subunit) were found. Further specification was obtained by MALDI-TOF MS detecting molecular ions at m/z 40109 and 19 896. These two fragments were also found on a western blot using an SDS-boiled chicory root extract and chicken-raised polyclonal antibodies against the purified 1-SST, indicating that the enzyme is a heterodimer in vivo. The N-terminus of chicory root 1-SST α-subunit was shown to be highly homologous with the cDNA-derived amino acid sequences from barley 6-SFT and a number of β-fructosyl hydrolases (in-vertases and fructan hydrolases). However, chicory root 1-SST properties could be clearly differentiated from those of chicory root 1-FFT (EC 2.4.1.100), chicory root acid invertase (EC 3.2.1.26) and yeast invertase. The enzyme mainly produced 1-kes-tose and glucose from physiologically relevant sucrose concentrations, indicating that this 1-SST is the key enzyme initiating fructan biosynthesis in vivo. However, like chicory root 1-FFT and barley 6-SFT, the enzyme also showed some β-fructofuranosi-dase activity (fructosyl transfer to water) at very low sucrose concentrations. Although sucrose clearly is the best substrate for the enzyme, some transferase and β-fructofuranosidase activity were also detected using 1-kestose as the sole substrate.