Immunoaffinity purification and characterization of leucine aminopeptidase from human liver.
Leucine aminopeptidase was purified from human liver cytosol to homogeneity, 1538-fold, with a yield of 84.4% by immunoaffinity chromatography. Increases in the activity and the stability of the enzyme were simultaneously observed during the purification procedure, suggesting the presence of some endogenous inhibitor in cytosol. The specific activity and Km value of the enzyme for L-leucine amide were found to be 58.00 mumol/min/mg of protein and 4.02 mM, respectively, at pH 8.0. The molecular weight of the enzyme was determined to be 360,000 by both polyacrylamide gradient gel electrophoresis and Sephadex G-200 gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of native and dimethyl suberimidate cross-linked enzyme indicate that the native enzyme has two subunits of Mr 53,000 (a) and 65,000 (b) and is a hexamer arranged as a trimer of dimers (3 X (a X b)). The optimum pH was 10.5, and the enzyme was stable in the pH range from 7.5-8.5. The enzyme was activated by divalent metal ions, especially by Mg2+ and Mn2+, with no change in Km value. The enzyme was inhibited by metal-chelating agents, indicating it to be a metalloenzyme. Amastatin and bestatin strongly inhibited the enzyme, but leupeptin did not. The enzyme had a broad substrate specificity toward oligopeptides and amino acid amides but had little or no activity toward chromogenic substrates. The enzyme also could hydrolyze natural substrates contained in liver cytosol and accordingly produce many kinds of amino acids commonly found in proteins.
D002413:Cations, Divalent; D002846:Chromatography, Affinity; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D007931:Leucyl Aminopeptidase / Q000302:isolation & purification* / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity
D002413:Cations, Divalent; D011480:Protease Inhibitors; D007931:Leucyl Aminopeptidase