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

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

Research output: Contribution to journalArticle

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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 - 1991
Externally publishedYes

Fingerprint

Liver
Purification
Taurine
Enzymes
Alanine
Glycine
High performance liquid chromatography
Bile Acids and Salts
Gel Chromatography
Gels
High Pressure Liquid Chromatography
Glycocholic Acid
Affinity chromatography
Agarose Chromatography
Antibodies
Isoelectric Point
Molecular mass
Chromatography
Electrophoresis
Affinity Chromatography

ASJC Scopus subject areas

  • Biochemistry

Cite this

Purification and characterization of bile acid-CoA : amino acid N-acyltransferase from human liver. / Johnson, M. R.; Barnes, S.; Kwakye, J. B.; Diasio, Robert B.

In: Journal of Biological Chemistry, Vol. 266, No. 16, 1991, p. 10227-10233.

Research output: Contribution to journalArticle

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