Karl J. Siegert

Quantification of adipokinetic hormones I and II in the corpora cardiaca of Schistocerca gregaria and Locusta migratoria

Abstract 1. 1. A method is described to quantify the amounts of adipokinetic hormones I and II (AKHI and AKHII) in the corpora cardiaca (CC) of Schistocerca gregaria and Locusta migratoria . These two hormones can be separated in a single reversed-phase high-performance liquid chromatography step with 0.1% trifluoroacetic acid and acetonitrile as the solvent system. The peaks were identified by their retention times with reference to synthetic and/or natural peptides. The peaks were detected at 206 nm, their area integrated and compared with a series of areas obtained for known amounts of synthetic AKHI chromatographed under the same conditions. 2. 2. The first quantification data for AKHI and AKHII are presented for 5th instar larvae and adult S. gregaria and L. migratoria 3. 3. At any given time during the period studied it was found that the CC always contained more AKHI than AKHII; the molar ratio AKHI: AKHII ranged from 2:1 to 6:1. 4. 4. S. gregaria contained more of the two peptides than L. migratoria . 5. 5. In most of the developmental stages studied more hormone was found in females than in males.

Elucidation of the primary structures of the cockroach hyperglycaemic hormones I and II using enzymatic techniques and gas-phase sequencing

ABSTRACT. Two peptides, HGHI and HGHII, both inducing hyperglycaemia and activation of fat body glycogen phosphorylase can be isolated from the corpora cardiaca of the American cockroach, Periplaneta americana, using high‐performance liquid chromatography. Both peptides are N‐terminally blocked by a pyroglutamate residue and are thus not available for sequencing methods using the Edman degradation as this technique requires a free N‐terminus. The blocked peptides were treated with pyroglutamate aminopeptidase to cleave the pyroglutamate residue, and the C‐terminus of each peptide is also blocked and neither molecule can be cleaved by carboxypeptidase A. The following sequences for hyperglycaemic hormones HGHI and HGHII have been revealed using gas‐phase sequencing.

Preliminary characterisation of locust diuretic peptide (DP-1) and another corpus cardiacum peptide (LCCP)

Abstract A simple two step HPLC purification protocol is described for one of the locust diuretic peptides (DP-1) and a second corpus cardiacum peptide (LCCP) of unknown function. DP-1 and LCCP are extracted from corpora cardiaca in 20% aqueous methanol (v/v), and applied to a high performance size-exclusion chromatography (HPSEC) column, from which DP-1 and LCCP co-elute in a fraction designated F7/8; this corresponds to a relative molecular mass of ca 6000–7000. Without sample concentration, F7/8 (2 ml) is applied directly onto a wide-pore reversed-phase HPLC column; LCCP and DP-1 are eluted in ca 12 and 20 min respectively on an increasing linear gradient of 0.5% min, starting at 30% acetonitrile. The diuretic activity of the DP-1 material was confirmed using the cAMP assay (Morgan and Mordue, 1985a, Insect Biochem. 15, 247–257). DP-1 and LCCP were isolated from ca 650 locusts using the above procedures for sequence analysis by gas-phase sequencing. The low amount of DP-1 purified allowed only a partial and fragmentary sequence to be determined; insufficient material was available for amino acid analysis. The higher amount of LCCP allowed a partial sequence determination of residues 1–40. The titre of DP-1 in locust CC is calculated to be 1.7–2.1 pmol/CC.

Carbohydrate and lipid metabolism during the last larval moult of the tobacco hornworm, Manduca sexta

Abstract. At 25°C and with a light regime of 17 h light and 7h dark, the last larval moult of the tobacco hornworm, Manduca sexta, lasts approximately 32 h, during which profound changes of metabolism were observed. At the onset of the moult, which coincides with the cessation of feeding, the proportion of active fat body glycogen phosphorylase increased from 10 (‐2h) to 25–30% (Oh). A biphasic pattern with peak activities of 45–50% after t – 12 h and again just prior to the shedding of the cuticle (32 h) was subsequently observed. Haemolymph trehalose concentration decreased significantly from c. 35 (Oh) to 20mM (8h), but then recovered to an intermediate level (30mM; 12h). After completion of the moult, the trehalose concentration was 35–40 mM. The haemolymph glucose level in feeding fourth instar larvae was 4–5 mM, but decreased sharply before the onset of the moult to c. 1 mM, followed by a slow 6‐fold increase over the next 20h. Prior to the shedding of the cuticle, the glucose level dropped again dramatically. The haemolymph lipid level increased slowly from an initial level of 1.2–1.4mg/ml during the early part of the moult, reaching a maximum of 1.8mg/ml after /= 16 h. Afterwards, a decrease of c. 50% was observed until ecdysis occurred. Oxygen consumption per animal decreased steadily from 30–35 μl/min pre‐moult by approximately 70% to c. 10 μl/min but started to increase about 5 h before the animals resumed feeding.

Isolation of hyperglycaemic peptides from the corpus cardiacum of the american cockroach, Periplaneta americana

Abstract Two peptides, HGHI and HGHII, have been isolated from the CC (corpus cardiacum) of the American cockroach, Periplaneta americana , both showing hyperglycaemic and phosphorylase activating potency when tested in adult cockroaches. The isolation procedure involved extraction of CC with 80% methanol and two steps of HPLC (high-performance liquid chromatography). In the first step extracted material was eluted in 0.1% TFA (trifluoroacetic acid) from a molecular size exclusion column. Estimates of molecular mass indicate that both peptides have a molecular mass of about 1000 daltons. In the second step reversed-phase HPLC utilizing a solvent system consisting of 0.1% TFA and acetonitrile was used; a gradient starting at 25% acetonitrile, with a slope 0.3% acetonitrile min −1 was run over 25 min. The first major peak, HGHI, eluted after 13.7 min and caused a strong hyperglycaemic response as well as strong activation of fat body glycogen phosphorylase when the equivalent of 0.1 of a pair was tested. For HGHII eluting after 21.3 min the hyperglycaemic effect as well as phosphorylase activation were only 50% of the response obtained for HGHI when the equivalent of 0.1 of a pair was injected.

Breakdown of locust adipokinetic hormone I by malpighian tubules of Schistocerca gregaria

Abstract The degradation of synthetic adipokinetic hormone I (AKHI) was studied using homogenates of the Malpighian tubules (MTs) from Schistocerca gregaria. Three major breakdown products, AKHI-1, AKHI-2 and AKHI-3, were found when the reaction mixture was subjected to reversed-phase high-performance liquid chromatography (RP-HPLC). At pH 7.5 AKHI-1 and AKHI-3 were found in small amounts only, however, at pH 8.0 large amounts could be detected. It was concluded that more than one degradative enzyme must be responsible for the complete breakdown of AKHI. Analysis of the products showed that AKHI-1 contained the AKHI residues 1–6 (pGlu-Leu-Asn-Phe-Thr-Pro), AKHI-2 only residue 8 (Trp) and AKHI-3 residues 8–10 (Trp-Gly-Thr-NH2). None of the breakdown products exhibited lipid-mobilizing activity when tested at doses of 20 pmol per locust.

Adipokinetic hormone and developmental changes of the response of fat body glycogen phosphorylase in Manduca sexta

Abstract The maximum percentage of active fat body glycogen phosphorylase which can be induced by the indigenous adipokinetic hormone (M-AKH) undergoes changes during the development of the tobacco hornworm, Manduca sexta. In intact fifth-instar larvae and pharate pupae a maximum of 60–80% can be achieved. Two picomoles of peptide were required in larvae weighing 2–3 g, pharate pupae and pupae; 20 pmol in larger larvae (5.5–6.5 g). In larvae 1 day before wandering (9–10 g), pupae and adults (both day 1 and day 2) 40% active phosphorylase was the maximum. Ligated abdomens from larvae weighing 9–10 g, however, showed a maximum phosphorylase activation of about 60%. The dilution of the injected peptide in different haemolymph volumes influences the results but cannot explain all the observed differences. Other parameters, e.g. presence of peptidases in the haemolymph and on the surface of tissues bathed in haemolymph or uptake of M-AKH into Malpighian tubules may undergo developmental changes which influence the residence time of the peptide in the haemolymph.

Degradation of adipokinetic hormones

Abstract Homogenates from various tissues of the desert locust, Schistocerca gregaria , produced identical patterns of breakdown products when incubated with the neuropeptide adipokinetic hormone I (AKH I). Comparison of disappearance of AKH I from the incubation medium of semi-isolated and isolated Malpighian tubules (MTs) and fat body pieces indicates that both tissues contain substantial capacities to take up and/or breakdown peptide. While the MTs may remove peptide circulating in the haemolymph, the fat body may metabolize peptide which binds to cell surface receptors. Locusta migratoria and Periplaneta americana MTs contain the same complement of degrading enzymes since they produced identical primary products. The initial proteolytic attack on AKH I took place through a post-proline cleaving enzyme (PPCE, residue 6). The use of the synthetic substrate benzyloxycarbonyl-Gly-Pro7-amino-4-methyl-coumarin confirmed the presence of PPCE. Adipokinetic hormone II from S. gregaria was cleaved between phenylalanine and serine (residues 4 and 5) indicating the action of a chymotrypsin-like endopeptidase. The synthetic substrates glutaryl-Phe-7AMC and N-succinyl-Ala-Phe-Lys-7AMC were not cleaved by MT homogenates; however, such homogenates degraded a tyrosine-containing substrate, N-benzoyl-Tyr ethyl ester. Chymotrypsin from bovine pancreas cleaved all the above substrates.

Regulation of fat body glycogen phosphorylase activity in adult Manduca sexta

Abstract Fat body glycogen phosphorylase was activated very slowly when adult tobacco hornworms, Manduca sexta , were starved. By contrast, chilling led to a significant activation of the enzyme within 2 h. This is a direct effect, since enzyme activation can also be induced in vitro . Inactivation of glycogen phosphorylase occured very rapidly in intact animals and in vitro showing that the fine-tuning of the glycogen breakdown/synthesis enzyme system favours glycogen synthesis in these animals. Activation of fat body glycogen phosphorylase in adult M. sexta at 0°C in vitro could be prevented by supplementing the saline with glucose; trehalose was ineffective. This effect of glucose was dose-dependent between 2 and 6mg/ml. The same action could not, however, be detected in larval fat body. When adult fat body was incubated at 0°C for up to 4h, no progressive activation of glycogen phosphorylase was observed in the absence of glucose suggesting that glucose is not the main regulator of enzyme activity. Glucose, however, may be a potent modulator of the glycogen phosphorylase/glycogen synthase system.

Strategies for the isolation of insect peptides

Two distinct diuretic peptides affect the rate of fluid secretion of the Malpighian tubules in Locusta. Diuretic peptide (DP 1) has a molecular mass ca. 4500 and acts via cyclic AMP: the second diuretic peptide (DP 2) has a molecular mass of ca. 1000 and operates via an unknown transduction mechanism; the possible involvement of cGMP, calcium mobilization and the breakdown of phosphoinositides has been investigated. Preliminary sequence data for DP 1 is reported and details are provided concerning the isolation and characterisation of a peptide which is present in large amounts in the storage lobes of the corpora cardiaca and co-chromatographs with DP 1 on high performance size-exclusion chromatography. Adipokinetic hormones (AKH) present in the corpora cardiaca of insects can regulate lipid metabolism. AKH occurs in two forms as AKH I and as AKH II: these hormones have been isolated and characterised in both Locusta and Schistocerca. AKH I is a decapeptide of the following structure: pGlu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-ThrNH2 and is identical in Locusta and Schistocerca. AKH II is different in these two insects: AKH II-L from Locusta is: pGlu-Leu-Asn-Phe-Ser-Ala-Gly-TrpNH2 and AKH II-S from Schistocerca is: pGlu-Leu-Asn-Phe-Ser-Thr-Gly-TrpNH2. These structures have been established using newly developed quick and simple procedures employing HPLC and gas-phase sequencing which enables full structures to be elucidated from 2-3 nmoles of peptide. Comparisons are made between the structures of AKH and related peptides isolated from insects and crustacea.