Purification and partial sequencing of the XL-I form of xenobiotic-metabolizing medium chain fatty acid:CoA ligase from bovine liver mitochondria, and its homology with the essential hypertension protein.
The XL-I form of xenobiotic-metabolizing medium-chain fatty acid:CoA ligase was purified to apparent homogeneity from bovine liver mitochondria. The procedure gave rise to a 435-fold increase in specific activity, with a yield of 12%. 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) which had an apparent molecular weight of 62 kDa. N-Terminal sequence analysis of the enzyme gave no sequence, which indicates a blocked N-terminus. To obtain sequence data, the enzyme was cleaved at methionine residues using CNBr. The resulting peptides were separated by SDS-PAGE. The cleavage pattern revealed two large peptides with molecular weights of ca. 10,000 and 12,000, plus several smaller peptides of lesser intensity. The 10 kDa and 12 kDa peptides were electroblotted onto Trans-Blot, and then sequenced directly from the blot. The N-terminal sequences of these two peptides are presented. When compared with known sequences it was discovered that these two peptides both have high homology with regions of the SA essential hypertension protein. This suggests a role for a carboxylic acid:CoA ligase in the control of high blood pressure.
Vessey
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DA
Department of Veterans' Affairs Medical Center, San Francisco, CA 94121, USA.
Kelley
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M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D003066:Coenzyme A Ligases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006973:Hypertension / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry; D008930:Mitochondria, Liver / Q000201:enzymology* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017421:Sequence Analysis; D015262:Xenobiotics / Q000378:metabolism*; D000098859:Long-Chain-Fatty-Acid-CoA Ligase
D000596:Amino Acids; D007527:Isoenzymes; D010446:Peptide Fragments; D011506:Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D015262:Xenobiotics; C081817:Acsm3 protein, rat; D003066:Coenzyme A Ligases; C107128:xenobiotic medium chain fatty acid - coenzyme A ligase; C090799:FAA2 protein, S cerevisiae; D000098859:Long-Chain-Fatty-Acid-CoA Ligase; D003488:Cyanogen Bromide