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-NH2) and AF2 (KHEYLRF-NH2), by cleavage of the Lys1-Asn2 and Lys1-His2 peptide bonds, respectively. AF2 (1.2 nmol of HEYLRF-NH2 formed min[-1] (mg protein[-1])) was hydrolysed 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 inhibited by the amastatin (IC50, 9.0 microM), leuhistin (IC50, 1.25 microM) but was insensitive to puromycin, indicating a similarity to mammalian aminopeptidase N. The enzyme was also inhibited by arphamenine B (IC50, 9.0 microM), (2S, 3R)-3-amino-2-hydroxy-4-(4-nitrophenyl)butanoyl-L-leucine (IC50, 8.0 microM), bestatin (IC50, 15.0 microM) and 1 mM 1-10 bis-phenanthroline. The detergent Triton X-100 solubilised enzyme had a pI of 5.0 and after 1000-fold purification by ion-exchange chromatography, appeared to have a Mr of around 240,000 by SDS-PAGE. The purified aminopeptidase had a Km of 534 microM for the hydrolysis of AF1 and cleaved Phe1 from FMRF-NH2, but was unable to hydrolyse DFMRF-NH2 or FDMRF-NH2. 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.
Sajid
M
M
Department of Pure and Applied Biology, University of Leeds, UK.
Isaac
R E
RE
Harrow
I D
ID
D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017165:Ascaris suum / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D002852:Chromatography, Ion Exchange; D003374:Coumarins / Q000378:metabolism; D006868:Hydrolysis; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000201:enzymology; D009479:Neuropeptides / Q000378:metabolism*; D010455:Peptides; D011480:Protease Inhibitors / Q000494:pharmacology; D012995:Solubility
D000900:Anti-Bacterial Agents; D003374:Coumarins; D009479:Neuropeptides; D010455:Peptides; D011480:Protease Inhibitors; C063461:AF1 neuropeptide; C073964:AF2 neuropeptide; C016977:amastatin; D000626:Aminopeptidases; C028743:7-amino-4-methylcoumarin