Vessey D A
Department of Veterans Affairs Medical Center, San Francisco, California 94121, USA.
Dig Dis Sci. 2001 Feb;46(2):438-42. doi: 10.1023/a:1005677521373.
The ATP-dependent activation of short- and medium-chain fatty acids to their respective CoA thioester adducts was investigated in the colonic mucosa from swine. Subcellular fractionation of a homogenate of the mucosa from the entire length of the colon revealed a predominantly mitochondrial localization for activity toward fatty acids ranging from propionate through laurate. These activities could be released from mitochondria in soluble form by freeze-thaw lysis. Purification of these activities revealed that they all appeared to reside with a single enzyme. This suggests that the entire colon contains a single form of medium-chain fatty acid:CoA ligase (MCFA:CoA ligase). The ligase also had activity toward benzoate and salicylate, although this activity was significantly lower than activity toward medium-chain fatty acids. The enzyme had the highest activity at Vmax with butyrate as substrate and had the lowest Km for octanoate. Butyrate and octanoate were mutually inhibitory. Activity toward both substrates was also efficiently inhibited by cyclohexane carboxylate. The molecular weight of the enzyme was estimated by gel filtration chromatography to be ca. 46,500. These data indicate that the colonic MCFA:CoA ligase is significantly different from the hepatic and kidney MCFA:CoA ligases.
研究了猪结肠黏膜中短链和中链脂肪酸依赖ATP激活生成各自辅酶A硫酯加合物的过程。对整个结肠黏膜匀浆进行亚细胞分级分离，结果显示，从丙酸盐到月桂酸盐的脂肪酸活性主要定位于线粒体。通过冻融裂解，这些活性可以以可溶形式从线粒体中释放出来。对这些活性进行纯化后发现，它们似乎都存在于单一酶中。这表明整个结肠含有单一形式的中链脂肪酸辅酶A连接酶（MCFA:CoA连接酶）。该连接酶对苯甲酸盐和水杨酸盐也有活性，不过其活性明显低于对中链脂肪酸的活性。以丁酸盐为底物时，该酶在Vmax时活性最高，对辛酸酯的Km值最低。丁酸盐和辛酸酯相互抑制。环己烷羧酸盐也能有效抑制对这两种底物的活性。通过凝胶过滤色谱法估计该酶的分子量约为46,500。这些数据表明，结肠MCFA:CoA连接酶与肝脏和肾脏的MCFA:CoA连接酶有显著差异。
