Abstract
The purification of xenobiotic/medium‐chain fatty acid:CoA ligases (XM‐ligases) from human liver mitochondria resulted in the isolation of two chromatographically separable forms (HXM‐A and HXM‐B). These two forms were purified to near homogeneity, cleaved with cyanogen bromide, the resulting peptides separated, and the N‐terminus of two of the peptides partially sequenced. Identical sequences were obtained for HXM‐A and HXM‐B for the two peptides. These sequences were used to design probes for screening a human liver cDNA library. This resulted in the isolation of two overlapping cDNAs. Using these sequences we were able to design PCR primers that resulted in the isolation of a full‐length cDNA from a human cDNA library. The cDNA contained 1731 bp of open reading frame and coded for a 64,230‐Da protein. This protein bears 56.2% amino acid homology to the MACS1 (medium‐chain acyl‐CoA synthetase) enzyme, 58.7% homology to the bovine XL‐III XM‐ligase, and 81.5% homology to the bovine XL‐I XM‐ligase. The cDNA could be expressed in COS cells, and the expressed enzyme had greater benzoate activity than phenylacetate activity, which is consistent with the known substrate specificity of HXM‐A. © 2003 Wiley Periodicals, Inc. J Biochem Mol Toxicol 17:1–6, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10056