Purification and characterization of bile acid-CoA:amino acid N-acyltransferase from human liver.
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-beta-alanine (a 5-fluorouracil catabolite), but not beta-alanine, as substrates. Kinetic studies revealed apparent Km values for taurine, 2-fluoro-beta-alanine, and glycine of 1.1, 2.2, and 5.8 mM, respectively, with corresponding Vmax values of 0.33, 0.19, and 0.77 mumol/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.
Johnson
M R
MR
Department of Pharmacology, University of Alabama, Birmingham 35294.
Barnes
S
S
Kwakye
J B
JB
Diasio
R B
RB
D000217:Acyltransferases / Q000737:chemistry / Q000302:isolation & purification*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001717:Birds; D015153:Blotting, Western; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology*; D051381:Rats; D013045:Species Specificity; D013696:Temperature
D000596:Amino Acids; D000217:Acyltransferases; C026953:bile acid-CoA amino acid N-acyltransferase