A cytosolic form of aminopeptidase P from Drosophila melanogaster: molecular cloning and characterization.
Using a functional genomic approach, we have identified and characterized a cytosolic form of aminopeptidase P from Drosophila melanogaster. This study represents the first characterization of an insect aminopeptidase P. The complete sequence of a 12.5 kbp genomic clone from D. melanogaster showed the presence of a 1,839 bp ORF, encoding a protein of 613 amino acids with a calculated molecular mass of 68.5 kDa. The deduced amino acid sequence was 48% identical and 66% similar to rat and human cytosolic aminopeptidase P. Amino acids important for catalytic activity and the metal binding ligands were found to be conserved between Drosophila AP-P and its mammalian homologues. The recombinant enzyme expressed in Escherichia coli hydrolyzed the amino terminal Xaa-Pro bond of substance P and bradykinin, revealing its functional identity. Further enzyme characterization showed the enzyme to be a manganese-dependent metallopeptidase. Immunoblot analysis showed that DAP-P is located exclusively in the cytosol and is temporally regulated during Drosophila development. In the adult fly, the protein could be detected in gut, testis and ovary, with a high level of expression in brain.
Kulkarni
Gauri V
GV
Molecular Biology Research Laboratory, Department of Zoology, University of Pune, Ganeshkhind, Pune-411007, Maharashtra, India.
Deobagkar
Deepti D
DD
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001920:Bradykinin / Q000378:metabolism; D001921:Brain / Q000201:enzymology; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology*; D004247:DNA / Q000032:analysis; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D006801:Humans; D008345:Manganese / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013373:Substance P / Q000378:metabolism*; D013379:Substrate Specificity
D013373:Substance P; D008345:Manganese; D004247:DNA; D000626:Aminopeptidases; C022750:X-Pro aminopeptidase; D008666:Metalloendopeptidases; D001920:Bradykinin