TY - JOUR
T1 - Metabolism of AF1 (KNEFIRF-NH2) in the nematode, Ascaris suum, by aminopeptidases, endopeptidase and deaminase enzymes
AU - Sajid, Mohammed
AU - Keating, Christopher
AU - Holden-Dye, Lindy
AU - Harrow, Ian D.
AU - Isaac, R.Elwyn
PY - 1996/1
Y1 - 1996/1
N2 - We have studied the metabolism and inactivation of AF1 (KNEFIRF-NH2) by membranes prepared from the locomotory muscle of Ascaris suum. FIRF-NH2, NEFIRF-NH2 and KNEFIRF were identified as three primary degradation products, resulting from the action of an endopeptidase, aminopeptidase and a deamidase, respectively. The endopeptidase resembled mammalian 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 inhibited by amastatin and bestatin but not by puromycin. The deamidation of AF1 was inhibited by phenylmethylsulfonyl fluoride, p-chloromercuricphenylsulfonate and mercuric chloride, indicating that the deamidase enzyme is a serine protease with a requirement for a free thiol group for activity. AF1 (1 μM) induces an increase in tension and an increase in the frequency and amplitude of spontaneous contractions of an A. suum muscle strip. None of the aforementioned AF1 metabolites (2–20 μM) 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
AB - We have studied the metabolism and inactivation of AF1 (KNEFIRF-NH2) by membranes prepared from the locomotory muscle of Ascaris suum. FIRF-NH2, NEFIRF-NH2 and KNEFIRF were identified as three primary degradation products, resulting from the action of an endopeptidase, aminopeptidase and a deamidase, respectively. The endopeptidase resembled mammalian 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 inhibited by amastatin and bestatin but not by puromycin. The deamidation of AF1 was inhibited by phenylmethylsulfonyl fluoride, p-chloromercuricphenylsulfonate and mercuric chloride, indicating that the deamidase enzyme is a serine protease with a requirement for a free thiol group for activity. AF1 (1 μM) induces an increase in tension and an increase in the frequency and amplitude of spontaneous contractions of an A. suum muscle strip. None of the aforementioned AF1 metabolites (2–20 μM) 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
U2 - 10.1016/0166-6851(95)02521-9
DO - 10.1016/0166-6851(95)02521-9
M3 - Article
SN - 1872-9428
VL - 75
SP - 159
EP - 168
JO - Molecular and Biochemical Parasitology
JF - Molecular and Biochemical Parasitology
IS - 2
ER -