ISOLATION AND CHARACTERIZATION OF MICROSOMAL ACYL-COA THIOESTERASE - A MEMBER OF THE RAT-LIVER MICROSOMAL CARBOXYLESTERASE MULTIGENE FAMILY

We have isolated and characterized an acyl-CoA thioesterase from rat l iver microsomes. The enzyme consists mainly of a monomer of 59 kDa. Ho wever, the final preparation was found to contain minor amounts of a t rimeric form of the protein. The enzyme was purified more than 85-fold from isolated microsomes and used for NH2-terminal sequence analysis and for analysis of peptides isolated after proteolytic digestion. The NH2-terminal sequence was unique but highly conserved compared to tho se of other carboxylesterases. Internal sequence data, covering almost 20% of the protein, showed high similarity to the deduced amino acid sequences from a cDNA encoding a carboxylesterase synthesized in the l iver and subsequently secreted to the blood [Alexson, S. E. H., Finlay , T. H., Hellman, U., Diczfalusy, U. & Eggertsen, G., unpublished resu lts] and nonspecific rat liver microsomal carboxylesterase with isoele ctric point of 6.1 [Robbi, M., Beaufay, H. & Octave, J.-N. (1990) Bioc hem. J. 269, 451 - 458], thus confirming earlier suggestions that this enzyme is a member of the microsomal carboxylesterase multigene famil y. The peptide sequences contained two of the four conserved cysteic a cid residues found in other carboxylesterases. Amino acid analysis ind icated that the protein contains five cysteine residues in contrast to most other described carboxylesterases which contain four highly cons erved cysteins. The purified protein was used for immunization and the antiserum was used to detect the protein as well as its trimeric form , which is a minor component, in isolated rat liver microsomes. The an tiserum recognized proteins of similar sizes in microsomes and 100000 X g supernatant prepared from hamster brown adipose tissue, a tissue k nown to contain very high activity of carboxylesterase, and to recogni ze carboxylesterases isolated from porcine and rabbit liver.