Molecular cloning and functional expression of rat liver cytosolic acetyl-CoA hydrolase

A cytosolic acetyl-CoA hydrolase (CACH) was purified from rat liver to homo geneity by a new method using Triton X-100 as a stabilizer. We digested the purified enzyme with an endopeptidase and determined the N-terminal amino- acid sequences of the two proteolytic fragments. From the sequence data, we designed probes for RT-PCR, and amplified CACH cDNA from rat liver mRNA. T he CACH cDNA contains a 1668-bp ORF encoding a protein of 556 amino-acid re sidues (62 017 Da). Recombinant expression of the cDNA in insect cells resu lted in overproduction of functional acetyl-CoA hydrolase with comparable a cyl-CoA chain-length specificity and Michaelis constant for acetyl-CoA to t hose of the native CACH. Database searching shows no homology to other know n proteins, but reveals high similarities to two mouse expressed sequence t ags (91% and 93% homology) and human mRNA for KIAA0707 hypothetical protein (50% homology) of unknown function.