Journal of Nematology 39(2):153–160. 2007. © The Society of Nematologists 2007. Characterization of Aminopeptidase in the Free-living Nematode Panagrellus redivivus: Subcellular Distribution and Possible Role in Neuropeptide Metabolism E. P. Masler Abstract: Aminopeptidase was detected in homogenates of the free-living nematode Panagrellus redivivus with the aminoacyl substrate L-alanine-4-nitroanilide. Subcellular distribution of activity was 80% soluble and 20% membrane-associated. Aminopep- tidases in the two fractions differed in affinity for Ala-4-NA, with Km’s of 0.65 mM (soluble) and 2.90 mM (membrane). Specific activities (units/mg) at pH 7.8, 27°C were 9.10 (soluble) and 14.30 (membrane). Each enzyme was competitively inhibited by amastatin (90% at 100 µM inhibitor, IC50 = 3.7 µM) and inhibited by puromycin (30% at 500 µM) and 1,10-phenanthroline (IC50’s:; 148 µM, soluble; 89 µM, membrane). Activity was restored by Zn++, with maximum recoveries of 50% (soluble) and 90% (mem- ∼ brane), each at 23 µM ZnCl2. Estimated molecular masses for each were 150 kDa. FMRFamide-like neuropeptides behaved as competitive inhibitors. Modification of the N-terminal F of FMRFamide weakened inhibition by 95%, suggesting that the N-terminus is essential for binding to the enzyme. Two nematode FMRFamides, APKPFIRFa and RNKFEFIRFa, were the most potent tested. This is the first biochemical characterization of aminopeptidase in a free-living nematode other than Caenorhabditis elegans and demon- strates the high selectivity of the P. redivivus enzymes for neuropeptide substrates. Key words: FMRFamide-like peptide, inhibitor; membrane, metallopeptidase, neuropeptide, protease Nematodes, like other eukaryotic organisms, depend reproduction (Day and Maule, 1999; Maule et al., 2002; upon proteolytic enzymes for the regulation of essential Rogers et al., 2003). developmental, metabolic and physiological events. Panagrellus redivivus Goodey is a bacteriophagous, Aminopeptidases are members of clans of proteases in- oviviparous, free-living soil nematode, being one of the volved in many of these diverse processes (Barrett et al., few that do not lay eggs, but hatch juveniles internally 2004), are present in both the cytoplasm and as mem- (Perry, 2002). It is gonochoristic, producing equal brane components, and comprise a wide variety of numbers of males and females, in contrast to the widely structures and activities (Taylor, 1993). studied hermaphrodite, Caenorhabditis elegans Maupas. In nematodes, aminopeptidases are associated with Panagrellus redivivus thus presents developmental con- intestinal digestion and nourishment for embryogen- trasts with other nematodes and provides useful com- esis (Joshua, 2001; Laurent et al., 2001; Brooks et al., parisons for studying basic physiological and biochemi- 2003), neuropeptide processing and signal attenuation cal mechanisms. Aminopeptidase and the digestion of (Sajid and Isaac, 1995; Sajid et al., 1996, 1997; Masler, peptides have been described in P. redivivus (Masler et 2002), molting (Hong and Bouvier, 1993; Rhoads et al., al., 2001; Masler, 2002). The present report character- 1997; Rhoads and Fetterer, 1998) and reproduction izes aminopeptidases in cytosolic and membrane frac- (Brooks et al., 2003; Lilley et al., 2005). Investigation of tions from P. redivivus and demonstrates substrate pref- the interactions of aminopeptidases with neuropep- erences among a variety of nematode and non- tides has intensified because of the growing interest in nematode neuropeptides. neuropeptides as targets for parasite control (Maule et MATERIALS AND METHODS al., 2002) and because of growing evidence for the pres- ence in nematodes of large neuropeptide families in- Animals and sample preparation: Panagrellus redivivus cluding the FMRFamide-related peptides (Li et al., was grown axenically in liquid medium (Chitwood et 1999; Kim and Li, 2004; McVeigh et al., 2005), insulins al., 1995), and 1-wk old cultures containing mixed (Pierce et al., 2001) and others (Nathoo et al., 2001). stages (approximately 1.3 µl worms/10 µl medium) The FMRFamide peptides have been the most widely were harvested. Nematodes were rinsed 5 times with studied group in nematodes because of their ubiquity autoclaved water using 10 to 15 ml water/ml of settled and their involvement in the regulation of muscle con- worms for each rinse, then stored at -80°C until use. traction, with its effects upon feeding, locomotion and Extracts were prepared by homogenizing frozen nema- todes in 10 volumes of autoclaved HPLC-grade water per 1 volume of nematodes using a methanol-washed ground glass homogenizer. Homogenates were centri- fuged (40,000g, 5°C), and the supernatants were col- Received for publication January 29, 2007. lected. Pellets were washed 3 times in water by vigorous Nematology Laboratory, Agricultural Research Service, U. S. Department of Agriculture, Beltsville, MD 20705 agitation, followed each time by centrifugation as Thanks are given to Dr. David P. Thompson, Pfizer Animal Health, for gen- above. Washes were pooled with the original superna- erously providing the nematode RFamides. Mention of trade names or com- mercial products in this publication is solely for the purpose of providing tant, and the pool was designated as the soluble frac- specific information and does not imply recommendation or endorsement by tion. Aliquots were dried in a SpeedVac (Savant Instru- the United States Department of Agriculture. E-mail: [email protected] ments, Farmingdale, NY) and stored at −20°C. Aliquots This paper was edited by Isgouhi Kaloshian. of washed pellets were dried and stored as above. 153 154 Journal of Nematology, Volume 39, No. 2, June 2007 Membranes were prepared by processing dried pel- DFMRFa, FLRFa, pEDFLRFa, pEGRFa, pESLRWa, lets using a Native Membrane Protein Extraction Kit YGGFMRRVa (Sigma), KHEYLRFa, KPNFLRFa, (Calbiochem, San Diego, CA) following the manufac- RNKFEFIRFa, KNEFIRFa and APKPKFIRFa (gifts of turer’s protocol for frozen tissue. The protocol was D. P. Thompson, Pfizer Animal Health, Kalamazoo, MI). modified to accommodate small processing volumes Molecular mass estimates: Native molecular mass of the (50–100 µl), and the proprietary solubilization and soluble enzyme was estimated by high-performance size wash buffers were tested in the enzyme assay and found exclusion chromatography (HP-SEC) using Progel not to interfere with activity. The extraction yielded 3000 size exclusion (300 x 7.8 mm) and Progel 3000 solubilized membrane proteins, designated as the guard (75 x 7.8 mm) columns (Phenomenex, Tor- membrane fraction. This fraction was used immediately rance, CA). Elution buffer was 100 mM Tris-HCl, pH or stored at 5°C overnight. Total proteins were esti- 7.8, with 300 mM NaCl. Flow rate was 0.5 ml/min. Frac- mated by microBCA assay (Pierce Chemical Co., Rock- tions were collected in 96-well plates and assayed di- ford, IL) following the manufacturer’s protocol. rectly. The column was calibrated using Dextran blue Protease assay and inhibitor screening: Aminopeptidase and protein markers ranging from 18 kDa to 240 kDa activity was measured using methods modified from (Sigma). Supplementary molecular size estimates were Masler et al. (2001). Reaction buffer used to prepare all done on both the soluble and membrane-associated reaction components was 100 mM Tris-HCl, pH 7.8. enzymes using non-denaturing PAGE. Test samples Reactions were initiated by the addition of the substrate were dissolved in PAGE sample buffer (10 mM Tris- L-alanine-4-nitroanilide (Ala-4-NA: Fluka/Sigma, St. HCl, pH 8.8, 0.0025% bromophenol blue, 10% glyc- Louis, MO) to enzyme (soluble or membrane) in a erol) and separated on gradient gels (4–20%; NOVEX/ 96-well microtiter plate (Corning Plastics, Corning, NY) Invitrogen, Carlsbad, CA) using pH 8.6 running buffer in a total reaction volume of 180 µl. Substrate concen- (24 mM Tris, 192 mM glycine). Following electropho- trations varied from 0.5 mM to 8 mM, depending upon resis, sample lanes were cut sequentially into 2.5 mm the experiment. Typical enzyme sample amounts were sections, and each was incubated overnight at 27°Cin 5.5 µg (soluble) and 6.4 µg (membrane)/reaction. 300 µl reaction buffer containing 8 mM Ala-4-NA. Ref- Progress of the reactions was monitored over time by erence marker proteins applied to adjacent lanes were increased absorbance (410 nm, 27°C) from the produc- thyroglobulin (669 kDa), ferritin (440 kDa), catalase tion of para-nitroaniline (pNA). A reference standard (232 kDa), lactate dehydrogenase (140 kDa) and albu- of pNA (Sigma) was determined by linear regression to min (67 kDa) (Amersham/Pharmacia, Piscataway, NJ). absorb 30.74 mAU/nMol at 410 nm. Using this value, 1 Data analysis: Data were analyzed by linear and non- Unit of P. redivivus aminopeptidase activity is defined as linear regressions using Excel (Microsoft, Redmond, 1 nMol pNA produced/min at 27°C. Kinetic character- WA), and means were compared by t-test using Sigma- istics (Vmax and Km) were determined using Line- Stat (Systat, Point Richmond, CA). weaver-Burk double reciprocal plots. pH tolerance was estimated by preparing 100 mM Tris-HCl buffers as a RESULTS pH series between 6.0 and 9.2. Samples and substrate were each prepared in the different buffers, and the Aminopeptidase activity was detected in both soluble assay was performed as described. and membrane fractions prepared from P. redivivus. Thermal tolerance of the enzymes was determined by Maximum specific activities (Units/mg total protein)