METABOLISM OF AF1 (KNEFIRF-NH2) IN THE NEMATODE, ASCARIS-SUUM, BY AMINOPEPTIDASE, ENDOPEPTIDASE AND DEAMIDASE ENZYMES

We have studied the metabolism and inactivation of AF1 (KNEFIRF-NH2) b y membranes prepared from the locomotory muscle of Ascaris suum. FIRF- NH2, NEFIRF-NH2 and KNEFIRF were identified as three primary degradati on products, resulting from the action of an endopeptidase, aminopepti dase and a deamidase, respectively. The endopeptidase resembled mammal ian neprilysin (NEP, endopeptidase 24.11) in that the enzyme activity was inhibited by phosphoramidon and thiorphan and that it cleaved AF1 on the amino side of phenylalanine. The aminopeptidase activity was in hibited by amastatin and bestatin but not by puromycin. The deamidatio n of AF1 was inhibited by phenylmethylsulfonyl fluoride, p-chloromercu ricphenylsulfonate and mercuric chloride, indicating that the deamidas e enzyme is a serine protease with a requirement for a free thiol grou p for activity. AF1 (1 mu M) induces an increase in tension and an inc rease in the frequency and amplitude of spontaneous contractions of an A. suum muscle strip. None of the aforementioned AF1 metabolites (2-2 0 mu M) retained biological activity in this bioassay, indicating that the endopeptidase, aminopeptidase and deamidase have the potential to terminate the action of AF1 on locomotory muscle of A. suum.