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 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.
Hosokawa
M
M
Laboratory of Biochemical Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan. masakiyo@p.chiba-u.ac.jp
Suzuki
K
K
Takahashi
D
D
Mori
M
M
Satoh
T
T
Chiba
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002265:Carboxylic Ester Hydrolases / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004285:Dogs; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D005810:Multigene Family; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D015262:Xenobiotics
D017931:DNA Primers; D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D015262:Xenobiotics; D002265:Carboxylic Ester Hydrolases; D013869:Thiolester Hydrolases; C428284:acyl-coA hydrolase D1