Open AccessMolecular cloning and functional expression of rat liver cytosolic acetyl‐CoA hydrolase
Author(s) -
Suematsu Naoya,
Okamoto Kazuki,
Shibata Kiyotaka,
Nakanishi Yoko,
Isohashi Fumihide
Publication year - 2001
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.2001.02162.x
Subject(s) - complementary dna , biology , microbiology and biotechnology , biochemistry , hydrolase , peptide sequence , homology (biology) , messenger rna , amino acid , enzyme , gene
A cytosolic acetyl‐CoA hydrolase (CACH) was purified from rat liver to homogeneity 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. The CACH cDNA contains a 1668‐bp ORF encoding a protein of 556 amino‐acid residues (62 017 Da). Recombinant expression of the cDNA in insect cells resulted in overproduction of functional acetyl‐CoA hydrolase with comparable acyl‐CoA chain‐length specificity and Michaelis constant for acetyl‐CoA to those of the native CACH. Database searching shows no homology to other known proteins, but reveals high similarities to two mouse expressed sequence tags (91% and 93% homology) and human mRNA for KIAA0707 hypothetical protein (50% homology) of unknown function.