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

Cottrell, G. S., Hooper, N. M. and Turner, A. J. (2000) Cloning, expression, and characterization of human cytosolic aminopeptidase P: a single manganese(II)-dependent enzyme. Biochemistry, 39 (49). pp. 15121-15128. ISSN 0006-2960

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To link to this item DOI: 10.1021/bi001585c

Abstract/Summary

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 clan 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 (RACE). The open reading frame encodes a protein of 623 amino acids with a calculated 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 migrated 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 hydrolyze 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 spectroscopy of AP-P expressed in E. coli revealed the presence of 1 mol of manganese/mol of protein and insignificant amounts of cobalt, iron, and zinc. The enzymatic activity of AP-P was promoted in the presence of Mn(II), and this activation was increased further by the addition of glutathione. The only other metal ion to cause slight activation of the enzyme was Co(II), with Ca(II), Cu(II), Mg(II), Ni(II), and Zn(II) all being inhibitory. Removal of the metal ion from the protein was achieved by treatment with 1,10-phenanthroline. The metal-free enzyme was reactivated by the addition of Mn(II) and, partially, by Fe(II). Neither Co(II) nor Zn(II) reactivated the metal-free 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 the metal ion used in vivo.

Item Type:Article
Refereed:Yes
Divisions:Faculty of Life Sciences > School of Chemistry, Food and Pharmacy > School of Pharmacy > Division of Pharmacology
No Reading authors. Back catalogue items
ID Code:30289
Uncontrolled Keywords:Amino Acid Sequence Aminopeptidases/antagonists & inhibitors/genetics/*metabolism Cations, Divalent Cloning, Molecular Cytosol/*enzymology Enzyme Activation Escherichia coli/genetics Humans *Manganese Metalloproteins/antagonists & inhibitors/genetics/*metabolism Molecular Sequence Data Recombinant Proteins/metabolism Sequence Analysis, DNA Sequence Homology, Amino Acid
Publisher:American Chemical Society

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