Gerd Gäde

Sequence analyses of adipokinetic hormones II from corpora cardiaca of Schistocerca nitans, Schistocerca gregaria, and Locusta migratoria by fast atom bombardment mass spectrometry

Structures of the second adipokinetic hormones (AKH IIs) from three locust species have been assigned by fast atom bombardment mass spectrometry. The AKH II hormone is identical in two Schistocerca species, S. nitans and S. gregaria, but is different in Locusta migratoria. Both AKH IIs are related to red pigment-concentrating hormone (RPCH) from prawns, Schistocerca AKH II being [Thr6]-RPCH and Locusta AKH II being [Ala6]-RPCH. Schistocerca AKH II is also bioactive in Locusta individuals.

The Revolution in Insect Neuropeptides Illustrated by the Adipokinetic Hormone/Red Pigment-Concentrating Hormone Family of Peptides

The last decade has seen a surge in the knowledge on primary structures of insect neuropeptides. Particularly successful were isolations and sequence determinations of more than 30 members of the adipokinetic hormone/red pigment-concentrating hormone (AKH/RPCH) family of peptides. This brief overview describes the techniques used to obtain data on purification and structure such as high performance liquid chromatography, Edman sequencing and mass spectrometry. Moreover, a short account on the precursors and on the multiple functions of the peptides of the AKH/RPCH family in various crustacean and insect species is given

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.

Primary sequence analysis by fast atom bombardment mass spectrometry of a peptide with adipokinetic activity from the corpora cardiaca of the cricket Gryllus bimaculatus

The octapeptide AKH-G, isolated from the cricket Gryllus bimaculatus, stimulates lipid mobilization in both the cricket itself and in migratory locusts. The structure of AKH-G has been assigned by fast atom bombardment mass spectrometry (FABMS) as pGlu-Val-Asn-Phe-Ser-Thr-Gly-Trp-NH2, and it is most closely related to AKH II-S from Schistocerca species, which has a Leu2 unit. Synthetic AKH-G, prepared by solid phase techniques, had the same bioactivity as the natural substance. FABMS measurements, including high-resolution and metastable studies, were made employing 400-900 pmole of sample.

Amino acid sequence of a hypertrehalosaemic neuropeptide from the corpus cardiacum of the cockroach, Nauphoeta cinerea

The hypertrehalosaemic factor from the cockroach Nauphoeta cinerea, a decapeptide, has been assigned the structure >Glu-Val-Asn-Phe-Ser-Pro-Gly-Trp-Gly-Thr-NH2. The structure was assigned from its high-resolution fast atom bombardment mass spectrum and metastable studies on the M + H ion and confirmed by solid phase synthesis.

Structural data on hypertrehalosaemic neuropeptides from Cryptocercus punctulatus and Therea petiveriana : how do they fit into the phylogeny of cockroaches?

Hypertrehalosaemic neuropeptides from the corpora cardiaca of the cockroaches Cryptocercus punctulatus and Therea petiveriana were structurally analysed to gather phylogenetic information independent from that provided by morphoanatomical data. Isolation of the peptides by liquid chromatography and structural elucidation by Edman degradation and mass spectrometry revealed an identical octapeptide for both species: pGlu–Leu–Asn–Phe–Ser–Pro–Asn–Trp–NH2. This peptide, denoted Tem–HrTH, was previously found in tenebrionid beetles and in the cockroach Polyphaga aegyptiaca. Using this information for phylogenetic analysis yielded a peptide tree that supports the previous morphoanatomical data and thus places the woodroach Cryptocercus inside the cockroach subfamily Polyphaginae.

The metabolic neuropeptides of the corpus cardiacum from the potato beetle and the American cockroach are identical.

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 Colorado potato beetle, Leptinotarsa decemlineata. The sequences of both peptides, designated Led-CC-I and Led-CC-II, were determined by pulsed-liquid phase sequencing employing Edman degradation after deblocking enzymatically the N-terminal pyroglutamate residue. The C-terminal of both peptides were blocked and neither molecule was cleaved by carboxypeptidase. Both peptides were found to be octapeptides; Led-CC-I has the primary structure pGlu-Val-Asn-Phe-Ser-Pro-Asn-Trp-NH2, and Led-CC-II has the primary sequence pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-NH2. These structures are identical to the two hypertrehalosaemic hormones from the American cockroach. Preliminary experiments show that the synthetic peptides are apparently involved in the control of amino acid metabolism during flight of the potato beetle.

The Sequence of Acheta Adipokinetic Hormone and the Variation in Corpus cardiacum Content and Hyperlipaemic Response with Age

The principle neuropeptide separated by reversed-phase liquid chromatography RP -HPLC from extracts of the corpora cardica of Acheta domesticus showed strong adipokinetic activity when injected into Acheta. The N -terminal pyroglutamate of the peptide was removed by enzymatic digestion, and the remaining peptide sequenced. The structure is identical to the peptide Grb -AKH previously described from the corpus cardiacum (CC) of Gryllus bimaculatus ( pGlu - Val - Asn - Phe - Ser - Thr - Gly - Trp - NH2). The ED50 was (0.8 pmol) and saturation was achieved with injection of 2 pmol of synthetic Grb-AKH . The time to maximum hyperlipaemic response was 90 -120 min. The response of the fat body to injected synthetic Grb- AKH doubled in 4 days during the last stadium , but was never greater than half the maximum response of the adult stage. The adult adipokinetic response doubled from the first to the fourth day then gradually declined through day 16. The increased AKH response was timecorrelated to fat storage in the larvae and to lipid diposits in the oocytes in the adult. Synthetic Grb -AKH activated glycogen phosphorylase in the fat body of Acheta. The amount of Grb- AKH present in the CC changed very little throughout the last larval stadium and through the first 9 days of the adult stage, averaging about 15 pmol/gland pair. A second peptide (a hexadecapeptide) was isolated from the CC of Acheta and sequenced. Its structure is identical to a putative diuretic hormone previously described in Acheta.

On the Release of the Three Locust (Locusta migratoria) Adipokinetic Hormones: Effect of Crustacean Cardioactive Peptide and Inhibition by Sugars

An existing test to monitor the rate of adipokinetic hormone release from the corpora cardiaca (C C) of Locusta migratoria in vitro was improved, so that a constant basal rate of release was achieved and the amount of released Lom-AKH-I, II and III could be quantified by HPLC . This test system was subsequently used to demonstrate that a small peptide, which has been found in a few insect species including L. migratoria, crustacean cardioactive peptide (CCAP), induces release of all three AKHs. Moreover, 80 mᴍ trehalose reduces CCAP-induced release of AKHs in vitro, and 160 mᴍ glucose reduces this release even further. Glucose also had a greater inhibitory effect than trehalose on the spontaneous release and inhibited the high potassium-stimulated release of AKH from the CC in vitro. Eighty mᴍ sucrose, on the other hand, had no effect on the release of AKH . The effect of trehalose and glucose could be due to their use as an energy source, with trehalose first having to be converted to glucose. Whatever the stimulus, the three AKHs are released in the same proportions as they are found in the CC, which in vivo would make Lom-AKH-I, the most abundant AKH, the major effector of the biological effects of AKHs in adult locusts

Glycogen phosphorylase in the fat body of two cockroach species, Periplaneta americana and Nauphoeta cinerea: isolation, partial characterization of three forms and activation by hypertrehalosaemic hormones.

The presence of endogenous phosphorylase kinase and phosphorylase phosphatase in crude extracts of fat bodies from the cockroaches Nauphoeta cinerea and Periplaneta americana is demonstrated in vitro by activation/inactivation of glycogen phosphorylase under appropriate conditions. Fractionation of fat body extracts of both cockroach species on an anion-exchange medium results in the elution of three peaks with phosphorylase activity. According to their AMP dependency these activity peaks are designated as phosphorylase b (inactive without AMP), phosphorylase ab (active without AMP, but several stimulated with AMP) and phosphorylase a (active without AMP). It is shown chromatographically that incubating crude extracts of fat bodies from both cockroaches, under conditions where the phosphorylase kinase is active, results in all phosphorylase b being converted to the ab- or a-form , whereas under conditions where the phosphorylase phosphatase is active all phophorylase a is converted to the ab- or b-form . Endogenous phosphorylase kinase of N. cinerea crude fat body extract can convert vertebrate phosphorylase b into the a-form , and, conversely, vertebrate muscle p hosphorylase kinase and phosphorylase phosphatase, respectively, are able to convert partially purified N. cinerea phosphorylase aborb and the ab- und a-form , respectively. In resting cockroaches most of the phosphorylase activity resides in the b-form and only a small fraction (10% ) in the a-form , whereas between 26% (N . cinerea) and 35% (P. americana) occurs in the ab-form . Injection of endogenous hypertrehalosaemic peptides into N. cinerea (the decapeptide Bld-HrTH ) or P. americana (the two octapeptides Pea-CAH -I and II) causes interconversion of phosphorylase; after injection, mainly (60% ) phosphorylase a is present, while 25% and 15% exists in the ab- und b-form , respectively. Purification of the three phosphorylase forms from N. cinerea is achieved by anion-exchange chromatography on DEAE-Sephacel followed by affinity chromatography on AMP-Sepharose. The final specific activities are 2.1, 6.9 and 27.2 U /mg protein for the a-, ab- und b-form . The molecular mass of the active molecules on gel filtration is between 173,000 and 177,000, and SDS gel electrophoresis reveals a subunit mass of 87,100, suggesting a homodimeric structure for all three form s. Kinetic studies show hyperbolic saturation curves for the substrates glycogen and Pi respectively, with Kᴍ-values of 0.021, 0.019 and 0.073% for glycogen and 8.3, 6.3 and 17.9 mᴍ for Pi (a-, ab- and b-form ). Phosphorylase a exhibits a more or less hyperbolic response to AMP and needs 70 |iM A M P for m axim al stim ulation. The kinetics for the ab- and b-form s are sigm oidal and maximal activities are displayed at about 3 mᴍ (half-maximum activation as calculated from Hill plots are 55 and 280 μᴍ for the ab- und b-form , respectively). Caffeine is a strong inhibitor of the b-form , but has only a slight inhibiting effect (10 -20 % ) on the ab- and a-form in the presence of AMP.