Proline-specific dipeptidyl peptidase activity in the cockroach brain and intestine: Partial characterization, distribution, and inactivation of tachykinin-related peptides

Proline-specific dipeptidyl peptidase (DPP TV) is an established enzyme kno wn to degrade neuropeptides and peptide hormones in vertebrate tissues. DPP TV cleaves peptides at the Pro(2) residue. Because several neuropeptides o f the cockroach Leucophaea maderae, such as LemTRP-1 (APSGFLGVRamide), are potential substrates for this peptidase, we investigated the occurrence of proline-specific DPP activity in cockroach tissues. Partly purified DPP act ivity was characterized from the brain and midgut of L. maderae by using Gl y-Pro-4-nitroanilide as a substrate. The highest activity was obtained from the membrane fraction of intestine; about 10 times less activity (per mill igram protein) was obtained from brain membranes. A smaller amount of solub le DPP activity could also be identified in both tissues. Gel chromatograph y of the solubilized intestinal DPP activity revealed a molecular mass of a bout 75 kDa. The enzyme had a pH optimum of 8.5. Diprotin A (Ile-Pro-Ile) w as an efficient competitive inhibitor of the cockroach DPP, whereas other k nown DPP inhibitors were found to be less potent. When incubated with human and cockroach DPP IV, the cleavage products of LemTRP-1 were AP and SGFLGV Ramide (des-AP-LemTRP-1) as determined by mass spectrometry of high-perform ance liquid chromatography (HPLC)-purified peptide fragments. The AP fragme nt was biologically inactive and the des-AP fragment had a drastically redu ced myostimulatory activity on the hindgut of L. maderae. The blowfly TRP c allitachykinin-I (CavTK-I; APTAFYGVRamide) was cleaved in two steps to des- AP-CavTK-I and desAPTA-CavTK-I, showing that cockroach DPP does not only li berate Xaa-Pro, but also Xaa-Ala dipeptides. The fragment desAPTA-CavTK-I w as completely inactive on the cockroach hindgut. To compare, LemTRP-3 and C avTK-II, which lack a Pro(2), were not cleaved by DPP IV. Enzyme histochemi stry for DPP IV was performed on cryostat sections of brain and intestine w ith Gly-Pro-4-methoxy-2-naphthylamide as the substrate and Fast Blue B as t he chromogen. Strong histochemical labeling was seen in specific neuropils of the brain such as the calyces of the mushroom bodies, the antennal glome ruli, and the central body. Also, the inner lining of the midgut (the perit rophic membrane) and the malpighian tubules were strongly labeled by reacti on product. In both the brain and intestine, the enzyme-histochemical react ion was inhibited by diprotin A. (C) 2000 Wiley-Liss, Inc.