Lee M J, Cusinato O, Luswata R, Wheeler C H, Goldsworthy G J
Department of Biology, Birkbeck College, University of London, UK.
Regul Pept. 1997 Mar 26;69(2):69-76. doi: 10.1016/s0167-0115(97)02130-7.
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.
为研究受体对N端变异的耐受性，合成了蝗虫脂肪动激素-I（AKH-I）的新型类似物，并在N端进行了修饰。合成了这样一些类似物：通过在焦谷氨酸（pGlu）1和亮氨酸（Leu）2之间（Gly1a-AKH-I）或亮氨酸2和天冬酰胺（Asn）3之间（Gly2a-AKH-I和Gly2ab-AKH-I）插入甘氨酸残基，使N端焦谷氨酰残基与分子其余部分的距离更远。其他带有N端延伸的修饰激素有：(Ahx)n-AKH-I（Ahx. 氨基己酸）；HPP(Ahx)n-AKH-I（HPP. 羟基苯丙酸）和Ac(Ahx)n-AKH-I（其中n = 0 - 3）。最后，用乙酰化和非乙酰化氨基酸取代pGlu1：谷氨酸（Glu）、脯氨酸（Pro）、丙氨酸（Ala）和酪氨酸（Tyr）。通过体内脂质动员试验和体外乙酸摄取试验测试了这些修饰对生物活性的影响。在pGlu1和Leu2之间插入甘氨酸比在Leu2和Asn3之间插入甘氨酸对AKH-I活性的降低幅度更大，这可能表明生物活性需要在pGlu附近有一个疏水残基。此外，Leu2的N端残基对于活性是必需的（即，[去pGlu]-AKH-I无活性），除非游离N端被乙酰化：Ac[去pGlu]-AKH-I有活性，但活性较低。随着N端远离分子其余部分的延伸增加，HPP(Ahx)0 - 3 - AKH-I、Ac(Ahx)1 - 3 - AKH-I和插入甘氨酸的类似物的活性持续降低。然而，未封闭的(Ahx)n - AKH-I类似物的活性并非如此，并且两种试验中的活性似乎与氨基己酸残基的数量无关。同样，虽然在两种试验中用Tyr、Pro、Ala或Glu取代pGlu1能保留激素活性，但所得类似物的乙酰化并没有使活性持续增加，实际上与未封闭的类似物相比，AcGlu1 - AKH-I的活性降低了。HPP1 - AKH-I是所测试的修饰肽中活性最强的，在体外试验中的活性与天然肽几乎相同。
