Cloning, expression, and characterization of human cytosolic aminopeptidase P: A single manganese(II)-dependent enzyme

The mammalian bradykinin-degrading enzyme aminopeptidase P (AP-P; E.C. 3.4. 11.9) is a metal-dependent enzyme and is a member of the peptidase dan MG. AP-P exists as membrane-bound and cytosolic forms, which represent distinct gene products. A partially truncated clone encoding the cytosolic form was obtained from a human pancreatic cDNA library and the 5' region containing the initiating Met was obtained by 5' rapid accumulation of cDNA ends (RAC E). The open reading frame encodes a protein of 623 amino acids with a calc ulated molecular mass of 69,886 Da. The full-length cDNA with a C-terminal hexahistidine tag was expressed in Escherichia coli and COS-1 cells and mig rated on SDS-PAGE with a molecular mass of 71 kDa. The expressed cytosolic AP-P hydrolyzed the X-Pro bond of bradykinin and substance P but did not hy drolyze Gly-Pro-hydroxyPro. Hydrolysis of bradykinin was inhibited by 1,10- phenanthroline and by the specific inhibitor of the membrane-bound form of mammalian AP-P, apstatin. Inductively coupled plasma atomic emission spectr oscopy of AP-P expressed in E. coli revealed the presence of 1 mel of manga nese/mol of protein and insignificant amounts of cobalt, iron, and zinc. Th e enzymatic activity of AP-P was promoted in the presence of Mn(II), and th is activation was increased further by the addition of glutathione. The onl y other metal ion to cause slight activation of the enzyme was Co(II), with Ca(II), Cu(II), Mg(II), Ni(II), and Zn(LI) all being inhibitory. Removal o f the metal ion from the protein was achieved by treatment with 1,10-phenan throline. The metal-free enzyme was reactivated by the addition of Mn(II) a nd, partially, by Fe(II). Neither Co(II) nor Zn(II) reactivated the metal-f ree enzyme. On the basis of these data we propose that human cytosolic AP-P is a single metal ion-dependent enzyme and that manganese is most likely t he metal ion used in vivo.