Identification of di-(2-ethylhexyl) phthalate-induced carboxylesterase 1 in C57BL/6 mouse liver microsomes: purification, cDNA cloning, and baculovirus-mediated expression.
Several mouse carboxylesterase (CES) isozymes have been identified, but information about their roles in drug metabolism is limited. In this study, we purified and characterized a mouse CES1 isozyme that was induced by di-(2-ethylhexyl) phthalate. Purified mouse CES1 shared some biological characteristics with other CES isozymes, such as molecular weight of a subunit and isoelectronic point. In addition, purified mouse CES1 behaved as a trimer, a specific characteristic of CES1A subfamily isozymes. The purified enzyme possessed temocapril hydrolase activity, and it was found to contribute significantly to temocapril hydrolase activity in mouse liver microsomes. To identify the nucleotide sequences coding mouse CES1, antibody screening of a cDNA library was performed. The deduced amino acid sequence of the obtained cDNA, mCES1, exhibited striking similarity to those of CES1A isozymes. When expressed in Sf9 cells, recombinant mCES1 showed hydrolytic activity toward temocapril, as did purified mouse CES1. Based on these results, together with the findings that recombinant mouse CES1 had the same molecular weight of a subunit, the same isoelectronic point, and the same native protein mass as those of purified mouse CES1, it was concluded that mCES1 encoded mouse CES1. Furthermore, tissue expression profiles of mCES1 were found to be very similar to those of the human CES1 isozyme. This finding, together with our other results, suggests that mCES1 shares many biological properties with the human CES1 isozyme. The present study has provided useful information for study of metabolism and disposition of ester-prodrugs as well as ester-drugs.
Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan.
Tomomi
T
Furihata
Masakiyo
M
Hosokawa
Nao
N
Koyano
Takahiro
T
Nakamura
Tetsuo
T
Satoh
Kan
K
Chiba
D000595:Amino Acid Sequence; D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000096:biosynthesis* / Q000235:genetics; D003001:Cloning, Molecular / Q000187:drug effects / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification*; D004790:Enzyme Induction / Q000187:drug effects / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008862:Microsomes, Liver / Q000187:drug effects* / Q000201:enzymology; D008969:Molecular Sequence Data; D010795:Phthalic Acids / Q000494:pharmacology*; D010802:Phylogeny
D018076:DNA, Complementary; D010795:Phthalic Acids; C053316:di-(2-ethylhexyl) terephthalate; D002265:Carboxylic Ester Hydrolases; C474609:CES1 protein, human