ISOLATION OF A NEUROPEPTIDE-DEGRADING ENDOPEPTIDASE FROM THE LEECH THEROMYZON TESSULATUM

Extracts of head parts prepared from the leech Theromyzon tessulatum hydrolyse the Gly3-Phe4 bond of synthetic [D-Ala2, Leu5]enkephalin and the Gly-His bond of benzoyl-Gly-His-Leu. The metabolism of benzoyl-Gly-His-Leu was completely inhibited by captopril, consistent with an angiotensin-converting enzyme activity. Such an enzyme has recently been isolated from T. tessulatum. However, the enkephalin hydrolysis by captopril (100 mu M) was inhibited to a maximum of 70%. The residual activity hydrolyzing enkephalin was inhibited by phosphoramidon, consistent with the presence of endopeptidase-24.11, a mammalian enzyme implicated in the metabolism of neuropeptides. This enzyme was isolated using four steps of purification including gel-permeation and anion-exchange chromatographies followed by reverse-phase HPLC. This neuropeptide endopeptidase (of approximate molecular mass 45 kDa) hydrolyses, at pH 7 and 37 degrees C, both the Gly3-Phe4 bond of synthetic [D-Ala2, Leu5]enkephalin and the Phe8-His9 bond of angiotensin I. Cleavage of [D-Ala2, Leu5]enkephalin yields, respectively, the Tyr-D-Ala-Gly and Phe-Leu peptides with a specific activity of 29 nmol Tyr-D-Ala-Gly min(-1).mg protein(-1) (K-m 95 mu M). The hydrolysis of angiotensin I yields angiotensin II and the dipeptide His-Leu with a specific activity of 1.2. nmol angiotensin min(-1).mg protein(-1) (K-m 330 mu M). The metabolism of these peptides was totally inhibited by phosphoramidon. This study therefore provides biochemical evidence for neuropeptide-degrading endopeptidases in leeches.