TY - JOUR
T1 - Glycine and taurine conjugation of bile acids by a single enzyme
T2 - Molecular cloning and expression of human liver bile acid CoA:Amino acid N-acyltransferase
AU - Falany, Charles N.
AU - Johnson, Martin R.
AU - Barnes, Stephen
AU - Diasio, Robert B.
N1 - Copyright:
Copyright 2004 Elsevier B.V., All rights reserved.
PY - 1994/7/29
Y1 - 1994/7/29
N2 - In order to establish whether a single enzyme in human liver was capable of conjugating bile acids with both glycine and taurine, a cDNA encoding human liver bile acid-CoA:amino acid N-acyltransferase (hBAT) has been isolated and characterized. A specific immunoaffinity-purified rabbit anti-hBAT polyclonal antibody was used to screen a λZap XR human liver cDNA library resulting in the isolation of two unique clones. hBAT8 and hBAT9 (1669 and 1491 base pairs in length, respectively) were isolated following screening of 4 × 105 clones of the cDNA library. Restriction mapping and sequence analysis demonstrated that the cDNAs were identical except hBAT8 contained an additional 178 bases of 5′ sequence; hBAT8 was completely sequenced, characterized, and used for all subsequent studies. hBAT8 consisted of a 184-nucleotide 5′-nontranslated region, an open reading frame of 1,254 bases predicting a protein of 418 amino acids with a molecular mass of 46,296 Da, and a 3′-nontranslated region of 209 nucleotides followed by a poly(A)+ tail. The identity of the cDNA was confirmed by the following findings: 1) the open reading frame began with an ATG codon and was followed by a nucleotide sequence which, when translated, corresponded exactly to the first 17 NH2-terminal amino acids of purified human liver BAT; 2) cytosol of Escherichia coli XL1-Blue cells transfected with hBAT8 subcloned into an expression vector, pKK233-2, demonstrated significant enzymatic activity for the conjugation of both taurine and glycine with cholic acid; 3) bacterial expression of hBAT8 generated a protein that comigrated with hBAT from human liver during SDS-polyacrylamide gel electrophoresis and cross-reacted with a specific polyclonal rabbit anti-hBAT antibody during immunoblot analysis; 4) kinetic characteristics of the expressed enzyme were very similar to those reported for purified liver BAT. These data demonstrate that a single cDNAis present in human liver which codes for a protein capable of catalyzing the conjugation of cholic acid with both glycine and taurine.
AB - In order to establish whether a single enzyme in human liver was capable of conjugating bile acids with both glycine and taurine, a cDNA encoding human liver bile acid-CoA:amino acid N-acyltransferase (hBAT) has been isolated and characterized. A specific immunoaffinity-purified rabbit anti-hBAT polyclonal antibody was used to screen a λZap XR human liver cDNA library resulting in the isolation of two unique clones. hBAT8 and hBAT9 (1669 and 1491 base pairs in length, respectively) were isolated following screening of 4 × 105 clones of the cDNA library. Restriction mapping and sequence analysis demonstrated that the cDNAs were identical except hBAT8 contained an additional 178 bases of 5′ sequence; hBAT8 was completely sequenced, characterized, and used for all subsequent studies. hBAT8 consisted of a 184-nucleotide 5′-nontranslated region, an open reading frame of 1,254 bases predicting a protein of 418 amino acids with a molecular mass of 46,296 Da, and a 3′-nontranslated region of 209 nucleotides followed by a poly(A)+ tail. The identity of the cDNA was confirmed by the following findings: 1) the open reading frame began with an ATG codon and was followed by a nucleotide sequence which, when translated, corresponded exactly to the first 17 NH2-terminal amino acids of purified human liver BAT; 2) cytosol of Escherichia coli XL1-Blue cells transfected with hBAT8 subcloned into an expression vector, pKK233-2, demonstrated significant enzymatic activity for the conjugation of both taurine and glycine with cholic acid; 3) bacterial expression of hBAT8 generated a protein that comigrated with hBAT from human liver during SDS-polyacrylamide gel electrophoresis and cross-reacted with a specific polyclonal rabbit anti-hBAT antibody during immunoblot analysis; 4) kinetic characteristics of the expressed enzyme were very similar to those reported for purified liver BAT. These data demonstrate that a single cDNAis present in human liver which codes for a protein capable of catalyzing the conjugation of cholic acid with both glycine and taurine.
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M3 - Article
C2 - 8034703
AN - SCOPUS:0028108409
SN - 0021-9258
VL - 269
SP - 19375
EP - 19379
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 30
ER -