Purification and characterization of amino-acid N-choloyltransferase from human liver.

Amino-acid N-choloyltransferase was purified from human liver. The procedure resulted in about 100-fold enriched activity and glycine- and taurine-dependent activities co-purified and did not separate to any extent in any of the steps. The final enzyme preparation had an apparent molecular mass of 100 kDa by gel filtration, 118 kDa by sucrose density gradient centrifugation and was composed of two identical subunits which had a molecular mass of 52 kDa, as judged by dodecyl sulfate polyacrylamide disc gel electrophoresis. The glycine- and taurine-dependent activities showed optima at pH 7.2 and pH 6.5, respectively. Apparent Km values of purified enzyme were 3.2 and 0.6mM for glycine and taurine, respectively. The Km value for choloyl-CoA was 50 microM for the glycine-dependent activity and 87 microM for the taurine-dependent activity. Bile acid derivatives and cholesterol had an inhibitory effect on both glycine- and taurine-dependent activity in vitro; on the other hand, the reaction was stimulated by the addition of glutathione, EDTA and L-cysteine. Amino acid substrate specificity was restricted to glycine, taurine, beta-alanine and D-alpha-alanine. As well as choloyl-CoA, its deoxy derivatives were also good substrates for the enzyme.