Reaffirmation of the validity of enzymatic cleavage of lithocholic acid from N-epsilon-lithocholyl-L-lysine and N-alpha-CBZ-N-epsilon-lithocholyl-L-lysine.

Abstract

N-epsilon-lithocholyl-L-lysine or N-alpha-CBZ-N-epsilon-lithocholyl-L-lysine when incubated overnight at 37 degrees C with 3 K units of clostridial cholanoylaminoacid hydrolase (from Clostridium perfringens ATCC 19574) in the presence of disodium EDTA (0.1 M), beta-mercaptoethanol (0.1 M), and sodium acetate buffer, pH 5.6, released free lithocholic acid. The latter material was isolated by thin-layer chromatography and identified by combined gas-liquid chromatography-mass spectrometry in the full scan and selected-ion mode. In order to maintain its activity, the enzyme was always stored in 1.0-ml aliquots at temperatures below -20 degrees C and each aliquot when thawed was used immediately; any left over enzyme was never reused. Contrary to the observations of Yanagisawa et al. (J. Lipid Res. 1984. 25: 1263-1271) the results of this study reaffirm the validity of the original observations on the enzymatic cleavage of lithocholic acid from tissue-bound form.