N-terminal modifications to AKH-I from Locusta migratoria: assessment of biological potencies in vivo and in vitro.

To investigate the receptor tolerances to N-terminal variation, novel analogues to Locusta AKH-I (adipokinetic hormone) have been synthesized with modifications at the N-terminus. Analogues were made where the N-terminal pyroglutamyl residue was spaced further from the remainder of the molecule by the insertion of glycine residues between either pGlu1 and Leu2 (Gly1a-AKH-I, or Leu2 and Asn3 (Gly2a-AKH-I and Gly2ab-AKH-I). Other modified hormones with N-terminal extensions were: (Ahx)n-AKH-I (Ahx. aminohexanoic acid); HPP(Ahx)n-AKH-I (HPP. hydroxyphenyl propionate) and Ac(Ahx)n-AKH-I (where n = 0-3). Finally, acetylated and non-acetylated amino acids were substituted for pGlu1: Glu, Pro, Ala and Tyr. The effects of these modifications on biological potency were tested in the lipid mobilization assay in vivo and acetate uptake assay in vitro. The potency of AKH-I was reduced much more by insertion of glycine between pGlu1 and Leu2, than between Leu2 and Asn3, perhaps suggesting that a hydrophobic residue is required adjacent to the pGlu for biological activity. In addition, a residue N-terminal to Leu2 is necessary for activity (i.e., [despGlu]-AKH-I is inactive) unless the free N-terminus is acetylated: Ac[despGlu]-AKH-I is active, but has low potency. The potencies of HPP(Ahx)0-3-AKH-I, Ac(Ahx)1-3-AKH-I and glycine-inserted analogues decreased consistently with increasing extension of the N-terminus away from the remainder of the molecule. However, potencies of the unblocked (Ahx)n-AKH-I analogues did not, and potency in either assay did not appear related to the number of aminohexanoic residues. Similarly, while hormonal activity was retained by substitution of pGlu1 by Tyr, Pro, Ala or Glu in both assays, acetylation of the resulting analogues did not provide a consistent increase in potency, but actually decreased for AcGlu1-AKH-I compared with its unblocked analogue. HPP1-AKH-I was the most potent of the modified peptides tested, with almost the same potency in the assay in vitro as the natural peptide.