Characterization of recombinant human adipocyte-derived leucine aminopeptidase expressed in Chinese hamster ovary cells

Adipocyte-derived leucine aminopeptidase (A-LAP) is a recently identified n ovel 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 th is study, recombinant A-LAP was expressed in Chinese hamster ovary cells, p urified to homogeneity and its enzymatic properties were characterized. The purified enzyme was active towards a synthetic substrate, L-leucyl-p-nitro anilide, yielding a V-max of 3.55 mu mol/min/mg and a K-m of 1.28 mM, and w as shown to be a monomeric protein with molecular mass of 120 kDa in soluti on, By monitoring the sequential N-terminal amino acid liberation, it was f ound that the enzyme hydrolyzes a variety of bioactive peptides, including angiotensin II and kallidin. Immunohistochemical analysis indicated that th e enzyme is expressed in the cortex of the human kidney, where tissue kalli krein is localized. Taken together, these results indicate that A-LAP posse sses a broad substrate specificity towards naturally occurring peptide horm ones and suggest that it plays a role in the regulation of blood pressure t hrough the inactivation of angiotensin II and/or the generation of bradykin in in the kidney.