MOLECULAR-CLONING AND EXPRESSION OF CDNA-ENCODING RAT-BRAIN CYTOSOLICACYL-COENZYME-A THIOESTER HYDROLASE

The cDNA encoding rat brain cytosolic acyl-CoA thioester hydrolase (AC T) has been cloned and sequenced, and the primary structure of the enz yme has been deduced. A partial amino acid sequence (38 amino acids) o f the enzyme was determined using the peptides generated after CNBr di gestion of the purified enzyme. Primers synthesized on the basis of th is information were used to isolate two cDNA clones, each encoding the full length of the enzyme. The nucleotide sequences of these clones c ontained an open reading frame encoding a 358-amino acid polypeptide w ith a calculated molecular mass of 39.7 kDa, similar to that determine d for the purified enzyme (40.9 kDa). The deduced ACT sequence showed no homology to the known sequences of any other thioesterases nor to a ny other known protein sequence. However, there was a strong homology to a number of expressed sequence tag human brain cDNA clones. The ide ntity of the ACT cDNA was confirmed by the expression of ACT activity in Escherichia coli. There was a 10-15-fold increase in ACT-specific a ctivity in the bacterial extracts after induction with isopropyl thio- galactoside, and the properties of the expressed enzyme (fusion protei n) were the same as those of the purified rat brain ACT. Northern blot analysis showed that a 1.65-kilobase ACT transcript was present in ra t brain and testis but not in any other rat tissues examined. However, the ACT mRNA was induced in the liver of rats that were fed Wy-14,643 , a peroxisome proliferator and inducer of rodent liver cytosolic acyl -CoA thioesterase. These results indicate that the induced rat liver A CT is homologous to the constitutive rat brain ACT.