Materials and methods Directory UMM :Data Elmu:jurnal:I:Insect Biochemistry and Molecular Biology:Vol30.Issue11.Nov2000:

1062 K.J. Siegert et al. Insect Biochemistry and Molecular Biology 30 2000 1061–1067 Table 1 Sequences of relevant AKH peptides including the novel AKH-III isolated in the present study. Molecular masses are given in brackets. Residues identical with those in Phm-AKH-III are given in bold type-face, those similar are italicised, e.g. leucine and isoleucine. The sequencing results of deblocked Phm-3 and Dis-3 are also given Pab-RPCH 929.4 pGlu Leu Asn Phe Ser Pro Gly TrpNH 2 Lom-AKH-I 1158.5 pGlu Leu Asn Phe Thr Pro Asn Trp Gly ThrNH 2 Phm-AKH-I 1144.5 pGlu Leu Asn Phe Thr Pro Asn Trp Gly SerNH 2 Lom-AKH-II 903.4 pGlu Leu Asn Phe Ser Ala Gly TrpNH 2 Scg-AKH-II 933.4 pGlu Leu Asn Phe Ser Thr Gly TrpNH 2 Lom-AKH-III 1072.5 pGlu Leu Asn Phe Thr Pro Trp TrpNH 2 Phm-AKH-III 1072.5 pGlu Ile Asn Phe Thr Pro Trp TrpNH 2 Cycle Residue Phm-3 Dis-3 pmol pmol 1 Ile 44.6l 35.7 2 Asn 44.2 32.9 3 Phe 42.3 31.4 4 Thr 27.2 19.8 5 Pro 19.1 13.8 6 Trp 5.2 4.5 7 Trp 3.4 2.7 8 void — —

2. Materials and methods

2.1. Insects Insects were either taken from a colony at the Depart- ment of Zoology, University of Cape Town, L. migratoria or were collected in the field ca. 100 km north of Cape Town near the Langebaan lagoon and kept in a constant temperature room at 25 ± 2 ° C with bran and oleander leaves ad libitum. 2.2. Extractions CC were dissected and transferred into Eppendorf tubes which were kept on crushed ice and contained either 50 µ l 0.1 trifluoroacetic acid TFA or 50 µ l 20 acetonitrile in 0.1 TFA. Tissues were then son- icated for 30 s on ice with a Branson sonicator, spun for 10 min 13,000 rpm and immediately used for reversed- phase high-performance liquid chromatography RP- HPLC. Other extracts were kept at 270 ° C until they were taken to Aberdeen for HPLC analysis. 2.3. RP-HPLC At the University of Cape Town, a Gilson system was used as described elsewhere Ga¨de, 1985 with an Ultra- sphere C-18 column 4.6 mm × 250 mm, plus guard car- tridge; Altex at a flow rate of 0.8 mlmin; 0.1 TFA pump A and acetonitrile pump B served as solvents. Peaks were detected in the ultraviolet range at l = 206 nm and also with a Shimadzu Model RF 535 fluor- escence HPLC monitor excitation wavelength 276 nm, emission wavelength 350 nm to identify tryptophan- containing peptides. The sensitivities of the spectropho- tometer and fluorometer were selected in a way that pep- tides containing one tryptophan residue had approxi- mately the same peak height in the two detection systems. The output was plotted on conventional chart recorders. Peaks were collected by hand into Eppendorf tubes, dried in vacuo and used for further HPLC analy- ses, mass spectrometry, bioassays or sequencing. Initially, a gradient that started at 20 acetonitrile was used; the percentage of B increased at a rate of 1 Bmin Gradient 1. AKHs were also separated with a gradient that started at 20 B but increased at 0.5 Bmin Gradient 2. For the separation of deblocked pep- tides the gradient started at 30 B and increased at 0.5 Bmin Gradient 3. CC extracts and collected peptides were also analysed at the University of Aberdeen using identical HPLC equipment. The same column was employed, however, a fluorometer was not available. Chromatograms were stored by the UniPoint software Version 1.40 which also controlled the HPLC pumps. Data were exported as text files, imported into Microsoft Excel Version 5.0a for the Power Macintosh and then copied into CA- Cricket Graph software Version 1.5 and plotted. 2.4. Bioassays Adipokinetic effects of isolated RP-HPLC fractions and synthetic peptides were determined by measuring changes in haemolymph lipid concentrations in adult locusts and D. spumans with the vanillin reagent Zo¨llner and Kirsch, 1962 as described elsewhere Ga¨de, 1980; changes in haemolymph carbohydrate 1063 K.J. Siegert et al. Insect Biochemistry and Molecular Biology 30 2000 1061–1067 concentrations were measured with the anthrone reagent Spik and Montreuil, 1964. 2.5. Deblocking of peptides Pyroglutamate aminopeptidase from Pyrococcus furiosus was used according to the manufacturer’s instructions Takara Shuzo. Peptides were dissolved in 25 µ l of the buffer 5 × diluted supplied with the enzyme and briefly sonicated. Enzyme 0.5 mU was added, the Eppendorf tube vortexed, spun and incubated for 2 h at 70 ° C with occasional vigorous agitation. Twenty-five microlitres of HPLC starting solvent 30 acetonitrile0.1 TFA was added and the mixture kept on ice until injection onto the RP-HPLC column Gradient 3. 2.6. Mass spectrometry Intact and deblocked peptides were analysed by mass spectrometry using a Voyager Elite matrix-assisted laser desorptionionisation time-of-flight instrument PerSeptive Biosystems. Samples were prepared in α - cyano-4-hydroxycinnamic acid and spectra acquired in positive, linear mode. 2.7. Peptide sequencing Deblocked peptides were subjected to automated Edman degradation using a model 477A sequencer con- nected to an on-line model 120 phenylhydantoin amino acid analyser both from Applied Biosystems. 2.8. Synthetic peptides Lom–AKH-III and Phm–AKH-III were synthesised by standard solid-phase chemistry, purified on RP- HPLC, and the identities verified by sequence determi- nation and mass spectrometry.

3. Results