Degradation of adipokinetic hormone family peptides by a circulating endopeptidase in the insect Manduca sexta.
The hemolymph (blood) of the Lepidopteran insect Manduca sexta contains an endopeptidase that metabolizes the nonapeptide Manduca adipokinetic hormone. In contrast to the situation in other insects, where the major site of inactivation is the Malpighian tubules (excretory organs), in Manduca the capacity of the hemolymph to metabolize adipokinetic hormone is comparable to that of the Malpighian tubules. The hemolymph enzyme cleaves Manduca adipokinetic hormone (pGlu-Leu-Thr-Phe-Thr-Ser-Ser-Trp-Gly-NH2) to give the fragment pGlu-Leu-Thr-Phe-Thr. Other fragments were not positively identified. The enzyme is present in the plasma and not in hemocytes, and occurs at similar levels in the hemolymph of larvae, pupae and adults. The enzyme is inactivated by boiling, has a neutral pH optimum (7.0-7.5), and an estimated molecular weight of 66 kDa. The enzyme was strongly inhibited by inhibitors of metalloprotease activity (EGTA and 1,10-phenanthroline), but not by serine protease inhibitors. The enzyme was capable of metabolizing a number of AKH family peptides with varying sequences around the presumed site of cleavage. An accurate assessment of enzyme kinetics was not possible with the assay method used, but the enzyme was not saturated at a substrate concentration of 10 microM, and the value of Km must be at least 1 microM. It is possible that the enzyme may represent a low affinity system of peptide removal rather than the principal means of inactivation.
Fox
A M
AM
School of Biological Sciences, University of Bath, Claverton Down, United Kingdom.
Reynolds
S E
SE
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D006458:Hemolymph / Q000201:enzymology; D007301:Insect Hormones / Q000378:metabolism*; D007700:Kinetics; D007814:Larva; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths / Q000201:enzymology* / Q000254:growth & development; D009842:Oligopeptides / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D007301:Insect Hormones; D009842:Oligopeptides; D011480:Protease Inhibitors; C044833:adipokinetic hormone; D010450:Endopeptidases; D011761:Pyrrolidonecarboxylic Acid