Puromycin-sensitive alanyl aminopeptidase from human liver cytosol: purification and characterization

Forensic Sci Int. 2000 Sep 11;113(1-3):143-6. doi: 10.1016/s0379-0738(00)00280-2.

Abstract

A cytosolic alanyl aminopeptidase (AAP-S) was purified to homogeneity from human liver cytosol. The molecular weight of the purified enzyme was calculated to be approximately 98,000 on TOF-MS and 90,000 on SDS-PAGE in the presence of beta-ME. These findings suggest that the enzyme exists as a monomeric form in human liver cytosol. The enzyme rapidly hydrolyzed the substrates Ala-, Lys- and Phe-MCAs, and moderately hydrolyzed Met-, Leu-, Tyr- and Lys-Ala-MCAs at pH ranging from 7.5 to 8.0. The order of the K(cat)/K(m) values of AAP-S at the optimal pH was Arg->Arg-Arg->Met->Leu->Lys->Phe->Lys-Ala->Tyr->Ala-MCAs. It was strongly inhibited by bestatin, leuhistin, actinonin, amastatin, 1, 10-phenanthroline, DFP, PCMBS, Zn(2+), Cd(2+), Co(2+), Cu((2+)), Hg(2+) and puromycin. AAP-S was approximately 80 times more sensitive than human seminal plasma AAP (aminopeptidase N, membrane type). The amino acid sequence of the first 60 residues of AAP-S was highly homologous with the N-terminal amino acid sequence of the rat liver puromycin-sensitive enkephalin-degrading aminopeptidase. These physicochemical properties and findings indicate that AAP-S from human liver cytosol is identical to those of other puromycin-sensitive aminopeptidase(s). Furthermore, with immunohistochemistry the enzyme was strongly stained in the cytoplasm of liver cells and renal tubules, and was ubiquitously localized in various human tissues.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aminopeptidases / chemistry
  • Animals
  • CD13 Antigens / analysis
  • CD13 Antigens / chemistry*
  • CD13 Antigens / isolation & purification*
  • CD13 Antigens / physiology
  • Cytosol / enzymology*
  • Electrophoresis, Polyacrylamide Gel
  • Humans
  • Hydrolysis
  • Immunohistochemistry
  • Liver / enzymology*
  • Molecular Sequence Data
  • Molecular Weight
  • Protein Synthesis Inhibitors*
  • Puromycin*
  • Rats
  • Sequence Homology, Amino Acid

Substances

  • Protein Synthesis Inhibitors
  • Puromycin
  • Aminopeptidases
  • enkephalin degrading enzyme
  • CD13 Antigens