Radioassay of bile acid coenzyme A: glycine/taurine: N-acyltransferase using an n-butanol solvent extraction procedure

Martin R. Johnson, Stephen Barnes, Robert B Diasio

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

A rapid, specific, and sensitive radioassay for measuring bile acid CoA:glycine/taurine: N-acyltransferase (EC 2.3.1) has been developed. In this assay, 3H-labeled amino acids (glycine or taurine) are conjugated with unlabeled bile acid CoA derivatives to form 3H-labeled bile acid amidates. Following incubation, the 3H-labeled bile acid amidate is separated from the unreacted amino acid by an n-butanol extraction method. The extraction procedure was developed by evaluating the effects of buffer concentration and pH on the recovery of radiolabeled bile acid amidate standards in the presence of human hepatic cytosol. Highest recovery (greater than 90%) of bile acid amidate standards occurred under acidic conditions (pH 2) in the presence of 1% ( w v) SDS. When the radioassay and accompanying n-butanol extraction procedure were utilized to study the amidation of glycine or taurine with cholic acid in human hepatic cytosol, a single peak of radioactivity corresponding with either authentic glycocholate or taurocholate was detected in the n-butanol phase by high-performance liquid chromatography. This assay for bile acid CoA:glycine/taurine: N-acyltransferase activity was linear with incubation time and protein concentration. This assay should be useful in the biochemical studies of this enzyme, as well as in the examination of bile acid amidation in clinical liver specimens.

Original languageEnglish (US)
Pages (from-to)360-365
Number of pages6
JournalAnalytical Biochemistry
Volume182
Issue number2
DOIs
StatePublished - Nov 1 1989
Externally publishedYes

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glycine acyltransferase
Acyltransferases
1-Butanol
Taurine
Solvent extraction
Coenzyme A
Bile Acids and Salts
Glycine
Assays
Cytosol
Liver
Glycocholic Acid
Cholic Acid
Amino Acids
Recovery
Taurocholic Acid
Radioactivity
High performance liquid chromatography
Buffers
High Pressure Liquid Chromatography

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Radioassay of bile acid coenzyme A : glycine/taurine: N-acyltransferase using an n-butanol solvent extraction procedure. / Johnson, Martin R.; Barnes, Stephen; Diasio, Robert B.

In: Analytical Biochemistry, Vol. 182, No. 2, 01.11.1989, p. 360-365.

Research output: Contribution to journalArticle

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