To clarify the reason for the high acyl-CoA hydrolase (ACH) activity found in dog liver microsomes, the ACH was purified to homogeneity using column chromatography. The purified enzyme, named ACH D1, exhibited a subunit molecular weight of 60 KDa. The amino terminal amino acid sequence showed a striking homology with rat liver carboxylesterase (CES) isozymes. ACH D1 possessed hydrolytic activities toward esters containing xenobiotics in addition to acyl-CoA thioesters, and these activities were inhibited by a specific inhibitor of CES or by CES RH1 antibodies. These findings suggest that this protein is a member of the CES multigene family. Since ACH D1 appears to be a protein belonging to the CES family, we cloned the cDNA from a dog liver lambdagt10 library with a CES-specific probe. The clone obtained, designated CES D1, possessed several motifs characterizing CES isozymes, and the deduced amino acid sequences were 100% identical with those of ACH D1 in the first 18 amino acid residues. When it was expressed in V79 cells, it showed high catalytic activities toward acyl-CoA thioesters. In addition, the characteristics of the expressed protein were identical with those of ACH D1 in many cases, suggesting that CES D1 encodes liver microsomal ACH D1.
Copyright 2001 Academic Press.