Purification and enzymatic properties of an l-leucine aminopeptidase from swine liver

Abstract

An l-leucine aminopeptidase (α-aminoacyl-peptide hydrolase (cytosol), EC 3.4.11.1), having a specificity toward the substrate l-leucine amide, but not toward l-leucyl β-naphthylamide or l-leucyl p-nitroanilide, has been purified 332-fold from swine liver, with a yield of 8.6%. This is the first purification of this enzyme from hepatic tissue. The purified enzyme submitted to analytical electrophoresis on cellulose acetate strips or in polyacrylamide gel showed a single band after staining with Ponceau S Red dye or Amido black, respectively. Purified swine liver l-leucine aminopeptidase, a cytosol enzyme, exhibited a molecular weight of 268 000 ± 50 000 by gel filtration. It hydrolyzed l-leucine amide substrate and l-leucyl peptides. It was activated by Mg 2+ and Mn 2+ and inhibited by Co 2+ and Zn 2+. The optimum pH was 10. It was rather sensitive to heat elevation. Swine liver l-leucine aminopeptidase was inhibited by EDTA, citric acid, isocaproic acid, dodecylamine, aliphatic alcohols and p-chloromercuribenzoate but unaffected by monoiodoacetic acid and diisopropyl fluorophosphate.