Isolation and sequencing of cDNAs for the XL-I and XL-III forms of bovine liver xenobiotic-metabolizing medium-chain fatty acid:CoA ligase.
The XL-I form of xenobiotic-metabolizing medium-chain fatty acid:CoA ligase was previously purified to apparent homogeneity from bovine liver mitochondria, and the amino acid sequence of a short segment of the enzyme was determined. This sequence was used to develop a probe for screening a bovine cDNA library from which a 1.6 kb cDNA was isolated. This cDNA was sequenced and found to contain the code for the known amino acid sequence. The complete open reading frame was not present in this cDNA, but it was estimated to code for approximately 75% of the XL-I sequence. The XL-III ligase was purified to apparent homogeneity from bovine liver mitochondria. The enzyme eluted from a gel filtration column as a single peak with an apparent molecular weight of ca. 55,000. It ran as a single band on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular weight of 62 kDa. N-Terminal sequence analysis of the enzyme gave no sequence, which indicates a blocked N-terminus. The enzyme was chemically cleaved using CNBr. The resulting peptides were separated by SDS-PAGE. The cleavage pattern revealed two large peptides of ca. 21 and 25 kDa, plus several smaller peptides including a prominent 6 kDa peptide. The N-terminus of the 6, 21, and 25 kDa peptides was sequenced and the 21 and 25 kDa sequences were identical indicating incomplete cleavage. The sequences were used to design probes for screening a bovine liver cDNA library. This resulted in the isolation of a 2,065 bp cDNA. This cDNA was sequenced and found to contain the initiation and termination codons, as well as the requisite amino acid sequences. The open reading frame coded for a 64,922 Da protein. The sequence of XL-III cDNA was markedly different from that of XL-I, indicating the genetic uniqueness of the two ligases. They are, however, 64% homologous, which suggests a common evolutionary origin.
Vessey
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Liver Study Unit, Department of Veterans' Affairs Medical Center San Francisco, CA 94121, USA.
Lau
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Kelley
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D003066:Coenzyme A Ligases / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D015262:Xenobiotics / Q000378:metabolism*
D018076:DNA, Complementary; D007527:Isoenzymes; D015262:Xenobiotics; D003066:Coenzyme A Ligases; C107128:xenobiotic medium chain fatty acid - coenzyme A ligase