Purification and characterization of bile acid-CoA:amino acid N-acyltransferase from human liver

M. R. Johnson, S. Barnes, J. B. Kwakye, R. B. Diasio

Research output: Contribution to journalArticle

38 Scopus citations

Abstract

The bile acid-conjugating enzyme, bile acid-CoA: amino acid N-acyltransferase, was purified 480-fold from the soluble fraction of homogenized frozen human liver. Purification was accomplished by a combination of anion exchange chromatography, chromatofocusing, glycocholate-AH-Sepharose affinity chromatography, and high performance liquid chromatography (HPLC) gel filtration. Following purification, the reduced, denatured enzyme migrated as a single 50-kDa protein band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A similar molecular mass was obtained for the native enzyme by HPLC gel filtration. Elution from the chromatofocusing column suggested an apparent isoelectric point of 6.0 (±0.2). Using a rabbit polyclonal antibody raised against the purified enzyme, Western blot analysis using 100,000 X g human liver supernatant confirmed that the affinity-purified polyclonal antibody was specific for human liver bile acid-CoA:amino acid N-acyltransferase. The purified enzyme utilized glycine, taurine, and 2-fluoro-β-alanine (a 5-fluorouracil catabolite), but not β-alanine, as substrates. Kinetic studies revealed apparent K(m) values for taurine, 2-fluoro-β-alanine, and glycine of 1.1, 2.2, and 5.8 mM, respectively, with corresponding V(max) values of 0.33, 0.19, and 0.77 μmol/min/mg protein. These data demonstrate that a single monomeric enzyme is responsible for the conjugation of bile acids with glycine or taurine in human liver.

Original languageEnglish (US)
Pages (from-to)10227-10233
Number of pages7
JournalJournal of Biological Chemistry
Volume266
Issue number16
StatePublished - Aug 16 1991

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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