PURIFICATION AND PARTIAL CHARACTERIZATION OF AN INDOMETHACIN HYDROLYZING ENZYME FROM PIG-LIVER

Purpose, Indomethacin is well known to be metabolized via O-demethylat ion and N-deacylation. In this paper we found an enzyme involved in th e hydrolysis of amide-linkage of indomethacin and partially characteri zed it as well as its substrate specificity. Methods, An indomethacin hydrolyzing enzyme was purified to homogeneity from pig liver microsom es using columns of Q-Sepharose, Red-Sepharose and Blue-Sepharose. The enzyme activity was assayed by measuring of rho-chlorobenzoic acid li berated from indomethacin by HPLC. Results. The purified enzyme effect ively hydrolyzed the amide linkage in indomethacin but not those in al pha-naphthylacetate and rho-nitrophenylacetate, which are typical subs trates for carboxylesterase. The subunit molecular mass of the enzyme was 65 kDa according SDS-polyacrylamide gel electrophoresis. The Micha elis constant (Km) and maximum velocity (Vmax) values for indomethacin were 67.8 mu M and 9.02 nmol/min/mg protein, respectively. The amino acid sequence analysis of the enzyme after cyanogen bromide cleavage s howed high homology with a mouse carboxylesterase isozyme designated a s ES-male. The activity of indomethacin hydrolysis was relatively high in the pig, rabbit and human liver homogenate, but not in those from rat and mouse. On the other hand purified human liver carboxylesterase s pl 5.3 and 4.5, and pig liver carboxylesterases have no catalytic ac tivity for indomethacin. Conclusions. These results indicate that the hydrolysis of amide-linkage of indomethacin in humans would be associa ted with an enzyme similar to the indomethacin hydrolyzing enzyme from pig liver microsomes described here.