title: Characterization of recombinant human adipocyte-derived leucine aminopeptidase expressed in Chinese hamster ovary cells.
abstract: Adipocyte-derived leucine aminopeptidase (A-LAP) is a recently identified novel member of the M1 family of zinc-metallopeptidases. Transfection of the A-LAP cDNA into COS-7 cells resulted in the secretion of the enzyme. In this study, recombinant A-LAP was expressed in Chinese hamster ovary cells, purified to homogeneity and its enzymatic properties were characterized. The purified enzyme was active towards a synthetic substrate, L-leucyl-p-nitroanilide, yielding a V(max) of 3.55 micromol/min/mg and a K(m) of 1.28 mM, and was shown to be a monomeric protein with molecular mass of 120 kDa in solution. By monitoring the sequential N-terminal amino acid liberation, it was found that the enzyme hydrolyzes a variety of bioactive peptides, including angiotensin II and kallidin. Immunohistochemical analysis indicated that the enzyme is expressed in the cortex of the human kidney, where tissue kallikrein is localized. Taken together, these results indicate that A-LAP possesses a broad substrate specificity towards naturally occurring peptide hormones and suggest that it plays a role in the regulation of blood pressure through the inactivation of angiotensin II and/or the generation of bradykinin in the kidney.
authors.affiliation: Laboratory of Cellular Biochemistry, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama 351-0198, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Hattori
authors.forename: K
authors.initials: K
authors.lastname: Kitatani
authors.forename: H
authors.initials: H
authors.lastname: Matsumoto
authors.forename: S
authors.initials: S
authors.lastname: Miyazawa
authors.forename: T
authors.initials: T
authors.lastname: Rogi
authors.forename: N
authors.initials: N
authors.lastname: Tsuruoka
authors.forename: S
authors.initials: S
authors.lastname: Mizutani
authors.forename: Y
authors.initials: Y
authors.lastname: Natori
authors.forename: M
authors.initials: M
authors.lastname: Tsujimoto
mesh_terms: D017667:Adipocytes / Q000201:enzymology*; D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D006801:Humans; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D007931:Leucyl Aminopeptidase / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D014162:Transfection
chemical_list: D000804:Angiotensin II; D007931:Leucyl Aminopeptidase