1998 Volume 13 Issue 4 Pages 405-417
Liver microsomal carboxylesterases (E. C. 3.1.1.1) function in the hydrolysis of a wide variety of endogenous and xenobiotic compounds, and play an important role in drug and lipid metabolism in many mammalian species. Multiple isozymes of liver microsomal carboxylesterases exist in various animal species, and the enzyme is involved in the metabolic activation of ester and amide-type prodrugs. In the present study, twenty-seven carboxylesterase isozymes were purified to electrophoretic homogeneity from liver microsomes of ten mammalian species and humans, and their physical, enzymological and immunological properties were compared with each other. And also we tried to compare the primary structure of carboxylesterase isozymes in various animal species and humans using cDNA cloning and analysis. The carboxylesterase isozymes from various species examined here showed considerable similarities in physicochemical and immunochemical properties, but not similar in substrate specificity. The deduced amino acid sequence of the clone possessed many structural characteristics that are highly conserved among rat RLl, RH1, RHlec, RS1, RS2, mouse ML1, MH1, MS1, guinea pig GP, beagle dog D1 and human HU1, including active site sequence (GESAGG, NKQEXG, GDHXD), and four cysteines that may be involved in the specific disulfide bond. It is well known that proteins that are retained in the endoplasmic reticulum (ER) lumen contain the retention signal at their carboxy terminal of the tetrapeptide (KDEL-COOH). The seven carboxylesterase clones (RL1, RH1, RHlec, MH1, GP, Dl and HUI) also contained an ER-retention signal of carboxylesterase (HXEL), and three clones (RS1, RS2, MS1) did not contain this peptide. When clone was expressed in COST and V79 cells, the plasmid-coded protein was retained. The cells expressing carboxylesterase is very high activity towards xenobiotic ester and amide. Site-specific mutation of the three amino acid residues of catalytic triad (Ser203 to Thr203, Glu335 to Ala 335, or His 448 to A1a448) greatly reduced the carboxylesterase activity. In conclusion, liver microsomal carboxylesterase in mammals and humans are closely involved in drug and lipid metabolism in the endoplasmic reticulum, and it is noteworthy that the isozymes from various species examined here showed considerable similarities in amino acid sequences, but not similar in substrate specificity. These reasons may be due to the variances of substrate binding site.
