Purification, characterization, and cloning of a cytosolic aspartyl aminopeptidase.
An aminopeptidase with a preference for N-terminal aspartyl and glutamyl residues but distinct from glutamyl aminopeptidase (EC 3.4. 11.7) was purified to near homogeneity from rabbit brain cytosol. Its properties were similar to an enzyme described previously (Kelly, J. A., Neidle, E. L., and Neidle, A. (1983) J. Neurochem. 40, 1727-1734). Aspartyl aminopeptidase had barely detectable activity toward simple aminoacyl-naphthylamide substrates. Its activity was determined with the substrate Asp-Ala-Pro-naphthylamide in the presence of excess dipeptidyl-peptidase IV (EC 3.4.14.5). The native enzyme has a molecular mass of 440 kDa and migrates as a single band of 55 kDa after SDS-polyacrylamide gel electrophoresis. The sequences of three tryptic peptides were used to screen the GenBankTM data base of expressed sequence tags. Human and mouse clones described as "similar to a yeast vacuolar aminopeptidase" and containing full-length cDNAs were identified and sequenced. The human cDNA was expressed in Escherichia coli. The amino acid sequence has significant homology to yeast aminopeptidase I, placing it as the first identified mammalian member of the M18 family of metalloproteinases. Homologous sequences in Caenorhabditis elegans and in prokaryotes revealed three conserved histidines, three conserved glutamates and five conserved aspartates. Aspartyl aminopeptidase is found at relatively high levels in all mammalian tissues examined and is likely to play an important role in intracellular protein and peptide metabolism.
Wilk
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Department of Pharmacology, Mount Sinai School of Medicine, New York, New York 10029, USA. S_Wilk@smtplink.mssm.edu
Wilk
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Magnusson
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D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology; D018819:Dipeptidyl Peptidase 4 / Q000378:metabolism; D004926:Escherichia coli; D043384:Glutamyl Aminopeptidase; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011817:Rabbits; D013379:Substrate Specificity
D000626:Aminopeptidases; D043384:Glutamyl Aminopeptidase; D018819:Dipeptidyl Peptidase 4