Purification, molecular cloning, and functional expression of dog liver microsomal acyl-CoA hydrolase: A member of the carboxylesterase multigene family

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 c hromatography. The purified enzyme, named ACH D1, exhibited a subunit molec ular weight of 60 KDa. The amino terminal amino acid sequence showed a stri king homology with rat liver carboxylesterase (CES) isozymes, ACH D1 posses sed hydrolytic activities toward esters containing xenobiotics in addition to acyl-CoA thioesters, and these activities were inhibited by a specific i nhibitor 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 lambda gt10 library with a CES-specific probe. The clone obtained, designa ted CES D1, possessed several motifs characterizing CES isozymes, and the d educed 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 showe d high catalytic activities toward acyl-CoA thioesters. In addition, the ch aracteristics of the expressed protein were identical with those of ACH D1 in many cases, suggesting that CES D1 encodes liver microsomal ACH D1. (C) 2001 Academic Press.