Characterization of Aminopeptidase in the Free-living Nematode Panagrellus redivivus: Subcellular Distribution and Possible Role in Neuropeptide Metabolism
Characterization of Aminopeptidase in the Free-living Nematode Panagrellus redivivus: Subcellular Distribution and Possible Role in Neuropeptide Metabolism
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作者：
M. Ep
以氨基基底物l -丙氨酸-4-硝基苯胺为底物，在自由生活的再病毒Panagrellus再病毒匀浆中检测到氨基肽酶。活性亚细胞分布80%为可溶性，20%为膜相关。两组氨基肽酶对Ala-4-NA的亲和力差异较大，分别为0.65 mM（可溶性）和2.90 mM（膜）。比活性（单位/mg）在pH 7.8、27℃时分别为9.10（可溶性）和14.30（膜）。每一种酶均被阿司他汀竞争性抑制（100 aeM时为90%，IC50 = 3.7 aeM），被嘌呤霉素（500 aeM时为30%）和1,10-菲罗啉（IC50's: 148 aeM，可溶；89 aeM，膜）抑制。ZnCl2在23 aeM下可恢复活性，最大回收率分别为50%（可溶性）和90%（膜）。估计分子质量为~ 150kda。fmrfamily样神经肽表现为竞争性抑制剂。修饰FMRFamide的n端F使抑制作用减弱了95%，这表明n端是与酶结合所必需的。两种线虫FMRFamides， APKPFIRFa和RNKFEFIRFa，是最有效的测试。这是除秀丽隐杆线虫外，第一次在自由生活的线虫中对氨基肽酶进行生化表征，并证明了再生线虫酶对神经肽底物的高选择性。
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. Aminopeptidases 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, 27oC were 9.10 (soluble) and 14.30 (membrane). Each enzyme was competitively inhibited by amastatin (90% at 100 aeM inhibitor, IC50 = 3.7 aeM) and inhibited by puromycin (30% at 500 aeM) and 1,10-phenanthroline (IC50's:; 148 aeM, soluble; 89 aeM, membrane). Activity was restored by Zn++, with maximum recoveries of 50% (soluble) and 90% (membrane), each at 23 aeM 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 demonstrates the high selectivity of the P. redivivus enzymes for neuropeptide substrates.