Vessey D A, Kelley M
Department of Veterans' Affairs Medical Center, San Francisco, CA 94121, USA.
Biochim Biophys Acta. 1997 Jun 23;1346(3):231-6. doi: 10.1016/s0005-2760(97)00038-6.
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.
辅酶A连接酶的XL-I形式。该纯化过程使比活性提高了435倍，产率为12%。该酶从凝胶过滤柱上洗脱下来时呈现为单一峰，表观分子量约为55,000。在SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）上它迁移为一条单一带，表观分子量为62 kDa。对该酶进行N端序列分析未得到序列，这表明N端被封闭。为了获得序列数据，使用溴化氰在甲硫氨酸残基处对该酶进行切割。所得肽段通过SDS-PAGE分离。切割模式显示出两条分子量约为10,000和12,000的大肽段，以及几条强度较小的较小肽段。将10 kDa和12 kDa的肽段电印迹到转印膜上，然后直接从印迹上进行测序。给出了这两条肽段的N端序列。与已知序列比较时发现，这两条肽段与SA原发性高血压蛋白的区域均具有高度同源性。这表明羧酸：辅酶A连接酶在高血压控制中发挥作用。
