In a search of the Caenorhabditis elegans DNA data base, an expressed sequence tag of 327 base pairs (termed cm01c7) with strong homology to the human leukotriene A 4 (LTA 4 ) hydrolase was found. The use of cm01c7 as a probe, together with conventional hybridization screening and anchored polymerase chain reaction techniques resulted in the cloning of the full-length 2.1 kilobase pair C. elegans LTA 4 hydrolase-like homologue, termed aminopeptidase-1 (AP-1). The AP-1 cDNA was expressed transiently as an epitope-tagged recombinant protein in COS-7 mammalian cells, purified using an anti-epitope antibody affinity resin, and tested for LTA 4 hydrolase and aminopeptidase activities. Despite the strong homology between the human LTA 4 hydrolase and C. elegans AP-1(63% similarity and 45% identity at the amino acid level), reverse-phase high pressure liquid chromatography and radioimmunoassay for LTB 4 production revealed the inability of the C. elegans AP-1 to use LTA 4 as a substrate. In contrast, the C. elegans AP-1 was an efficient aminopeptidase, as demonstrated by its ability to hydrolyze a variety of amino acid pnitroanilide derivatives. The aminopeptidase activity of C. elegans AP-1 resembled that of the human LTA 4 hydrolase/aminopeptidase enzyme with a preference for arginyl-p-nitroanilide as a substrate. Hydrolysis of the amide bond of arginyl-p-nitroanilide was inhibited by bestatin with an IC 50 of 2.6 ؎ 1.2 M. The bifunctionality of the mammalian LTA 4 hydrolase is still poorly understood, as the physiological substrate for its aminopeptidase activity is yet to be discovered. Our results support the idea that the enzyme originally functioned as an aminopeptidase in lower metazoa and then developed LTA 4 hydrolase activity in more evolved organisms.
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