Proline-specific dipeptidyl peptidase from the blue blowfly Calliphora vicina hydrolyzes in vitro the ecdysiostatic peptide trypsin-modulating oostatic factor (Neb-TMOF).
To elucidate the mechanisms of inactivation of the ecdysiostatic peptide trypsin-modulating oostatic factor (Neb-TMOF) in the blue blowfly Calliphora vicina, we investigated its proteolytic degradation. In homogenates and membrane and soluble fractions, this hexapeptide (sequence: NPTNLH) was hydrolyzed into two fragments, NP and TNLH, suggesting the involvement of a proline-specific dipeptidyl peptidase. The dipeptidyl peptidase activity was highest in the late larval stage. It was purified 240-fold from soluble fractions of pupae of mixed age and classified on the basis of several catalytic properties as an invertebrate homologue of mammalian dipeptidyl peptidase IV (EC 3.4.14.5). Fly dipeptidyl peptidase IV has a molecular mass of 200 kDa, showed a pH optimum of 7.5-8.0 with the chromogenic substrate Gly-Pro-4-nitroanilide, and cleaved other chromogenic substrates with penultimate Pro or, with lower activity, Ala. It liberated Xaa-Pro dipeptides from the N-terminus of several bioactive peptides including substance P, neuropeptide Y, and peptide YY but not from bradykinin, indicating that the peptide bond between the two proline residues was resistant to cleavage. Fly dipeptidyl peptidase belongs to the serine class of proteases as the mammalian enzyme does; the fly enzyme, however, is not inhibited by several selective or nonselective inhibitors of its mammalian counterpart. It is suggested that dipeptidyl peptidases exert a regulatory role for the clearance not only of TMOF in files but for other bioactive peptides in various invertebrates.
Martensen
I
I
Anatomical Institute, University of Kiel, Germany.
Koolman
J
J
Mentlein
R
R
D000818:Animals; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D018819:Dipeptidyl Peptidase 4 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004175:Diptera / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007301:Insect Hormones / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism*
D007301:Insect Hormones; D009842:Oligopeptides; C065612:Peptide oostatic hormone; D018819:Dipeptidyl Peptidase 4