Isolation and characterization of microsomal acyl-CoA thioesterase. A member of the rat liver microsomal carboxylesterase multi-gene family.
We have isolated and characterized an acyl-CoA thioesterase from rat liver microsomes. The enzyme consists mainly of a monomer of 59 kDa. However, the final preparation was found to contain minor amounts of a trimeric 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 those 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 liver and subsequently secreted to the blood [Alexson, S. E. H., Finlay, T. H., Hellman, U., Diczfalusy, U. & Eggertsen, G., unpublished results] and nonspecific rat liver microsomal carboxylesterase with isoelectric point of 6.1 [Robbi, M., Beaufay, H. & Octave, J.-N. (1990) Biochem. J. 269, 451-458], thus confirming earlier suggestions that this enzyme is a member of the microsomal carboxylesterase multigene family. The peptide sequences contained two of the four conserved cysteic acid residues found in other carboxylesterases. Amino acid analysis indicated that the protein contains five cysteine residues in contrast to most other described carboxylesterases which contain four highly conserved 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 antiserum recognized proteins of similar sizes in microsomes and 100,000 x g supernatant prepared from hamster brown adipose tissue, a tissue known to contain very high activity of carboxylesterase, and to recognize carboxylesterases isolated from porcine and rabbit liver.
Alexson
S E
SE
Department of Clinical Chemistry, Karolinska Institutet, Huddinge University Hospital, Sweden.
Mentlein
R
R
Wernstedt
C
C
Hellman
U
U
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D015153:Blotting, Western; D002265:Carboxylic Ester Hydrolases / Q000235:genetics*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D005810:Multigene Family; D010170:Palmitoyl-CoA Hydrolase / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D010446:Peptide Fragments / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D010446:Peptide Fragments; D002265:Carboxylic Ester Hydrolases; D010170:Palmitoyl-CoA Hydrolase; D014357:Trypsin