Purification and properties of a membrane aminopeptidase from Ascaris suum muscle that degrades neuropeptides AF1 and AF2

We have identified on the membranes of the locomotory muscle of Ascaris suum an amastatin-sensitive aminopeptidase that hydrolyses the bioactive neuropeptides AF1 (KNEFIRF–NH 2) and AF2 (KHEYLRF–NH 2), by cleavage of the Lys 1–Asn 2 and Lys 1–His 2 peptide bonds, respectively. AF2 (1.2 nmol of HEYLRF–NH 2 formed min −1 (mg protein −1)) was hydrolysed at a faster rate compared to AF1 (0.2 nmol of NEFIRF–NH 2 formed min −1 (mg protein −1)). AF1 hydrolysis by the aminopeptidase was inhibited by the amastatin (IC 50, 9.0 μM), leuhistin (IC 50, 1.25 μM) but was insensitive to puromycin, indicating a similarity to mammalian aminopeptidase N. The enzyme was also inhibited by arphamenine B (IC 50, 9.0 μM), (2 S, 3 R)-3-amino-2-hydroxy-4-(4-nitrophenyl)butanoyl- l-leucine (IC 50, 8.0 μM), bestatin (IC 50, 15.0 μM) and 1 mM 1-10 bis-phenanthroline. The detergent Triton X-100 solubilised enzyme had a p I of 5.0 and after 1000-fold purification by ion-exchange chromatography, appeared to have a M r of around 240 000 by SDS-PAGE. The purified aminopeptidase had a K m of 534 μM for the hydrolysis of AF1 and cleaved Phe 1 from FMRF–NH 2, but was unable to hydrolyse dFMRF–NH 2 or F dMRF–NH 2. The aminopeptidase that we have described in this report might have a role in the extracellular metabolism and inactivation of neuropeptides acting on the locomotory muscle of A. suum.