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 Ascari s suum an amastatin-sensitive aminopeptidase that hydrolyses the bioac tive neuropeptides AF1 (KNEFIRF-NH2) and AF2 (KHEYLRF-NH2), by cleavag e of the Lys(1)-Asn(2) and Lys(1)-His(2) peptide bonds, respectively. AF2 (1.2 nmol of HEYLRF-NH2 formed min(-1) (mg protein(-1))) was hydro lysed at a faster rate compared to AF1 (0.2 nmol of NEFIRF-NH2 formed min(-1) (mg protein(-1))). AF1 hydrolysis by the aminopeptidase was in hibited by the amastatin (IC50, 9.0 mu M), leuhistin (IC50, 1.25 mu M) but was insensitive to puromycin, indicating a similarity to mammalia n aminopeptidase N. The enzyme was also inhibited by arphamenine B (IC 50, 9.0 mu M), (2S, mino-2-hydroxy-4-(4-nitrophenyl)butanoyl-L-leucine (IC50, 8.0 mu M), bestatin (IC50, 15.0 mu M) and 1 mM 1-10 bis-phenan throline. The detergent Triton X-100 solubilised enzyme had a pI of 5. 0 and after 1000-fold purification by ion-exchange chromatography, app eared to have a M-r of around 240 000 by SDS-PAGE. The purified aminop eptidase had a K-m of 534 mu M for the hydrolysis of AF1 and cleaved P he(1) from FMRF-NH2, but was unable to hydrolyse DFMRF-NH2 or FDMRF-NH 2. The aminopeptidase that we have described in this report might have a role in the extracellular metabolism and inactivation of neuropepti des acting on the locomotory muscle of A. suum. (C) 1997 Elsevier Scie nce B.V.