Submitted to: Journal of Nematology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 1, 2007
Publication Date: June 1, 2007
Citation: Masler, E.P. 2007. Characterization of aminopeptidase in the free-living nematode Panagrellus redivivus: subcellular distribution and possible role in neuropeptide metabolism. Journal of Nematology. 39(2):153-160.
Interpretive Summary: Plant-parasitic nematodes attack all crops of agricultural importance, causing over $10 billion in losses annually to U.S. farmers. One problem facing growers is that environmental concerns will result in the elimination of the most extensively used chemical nematicide from the United States within the next few years. This loss makes the discovery of environmentally and economically sound replacement control agents critical. One approach to discovering new ways to control nematodes is to identify ways to disrupt their normal biochemical processes by using chemicals or enzymes that occur naturally inside the nematode. We have discovered an enzyme that degrades hormones controlling muscle function in a nematode that is not a plant parasite. The discovery can be used to identify similar enzymes in plant parasites which may lead to using the enzymes to control the nematodes. Consequently, this information will be used by researchers in the agrochemical and agricultural biotechnology industries who are developing safe, selective methods for nematode control.
Aminopeptidase was detected in homogenates of the free-living nematode Panagrellus redivivus with the aminoacyl substrate L-alanine-4-nitroanilide (Ala-4-NA). Subcellular distribution of the enzyme was unequal, with approximately 80 percent of total aminopeptidase in the soluble fraction and the remainder in the membranes. Aminopeptidases associated with the two fractions had different substrate affinities with Km = 0.65 ± 0.07 mM Ala-4-NA for the soluble enzyme and 2.90 ± 0.55 mM for the membrane form. Specific activities were 14.30 ± 1.63 Units/mg at pH 7.8, 27 degrees C for the membrane aminopeptidase and 9.10 ± 0.65 Units/mg for the soluble form. Each enzyme was competitively inhibited by amastatin (90 percent at 100 microM inhibitor, IC50 = 3.7 microM) but only weakly inhibited by puromycin (30 percent at 500 microM). The soluble aminopeptidase was inhibited by 1,10-phenanthroline (IC50 = 185 microM), with activity partially restored by Zn++. Estimated molecular weight (150kDa) was similar for the two enzymes. Neuropeptides in the FMRFamide-like peptide family behaved as competitive inhibitors of aminopeptidase. Modification of the N-terminal Phe of FMRFamide reduced inhibition by 95 percent, suggesting that the N-terminus is essential for binding to the enzyme. Two nematode neuropeptides, APKPFIRFa and RNKFEFIRFa, were the most potent peptides tested. This is the first biochemical characterization of aminopeptidase in a free-living nematode other than Caenorhabditis elegans and demonstrates the high selectivity of the P. redivivus enzymes for neuropeptide substrates.