Kasuya Fumiyo, Tatsuki Takao, Ohta Masayuki, Kawai Yuich, Igarashi Kazuo
Faculty of Pharmaceutical Sciences, Kobe-Gakuin University, Ikawadani, Nishiku, Japan.
Protein Expr Purif. 2006 Jun;47(2):405-14. doi: 10.1016/j.pep.2005.11.006. Epub 2005 Dec 5.
Medium chain acyl-CoA synthetases catalyze the first reaction of amino acid conjugation of many xenobiotic carboxylic acids and fatty acid metabolism. This paper reports studies on purification, characterization, and the partial amino acid sequence of mouse liver enzyme. The medium chain acyl-CoA synthetase was isolated from mouse liver mitochondria. The purified enzyme catalyzes this reaction not only for straight medium chain fatty acids but also for aromatic and arylacetic acids. Maximal activity was found with hexanoic acid. High activities were obtained with benzoic acid having methyl, pentyl, and methoxy groups in the para- or meta-positions of the benzene ring. However, the enzyme was less active with valproic acid and ketoprofen. Salicylic acid exhibited no activity. The medium chain acyl-CoA synthetases from mouse and bovine liver mitochondria were subjected to in-gel tryptic digestion, followed by LC-MS/MS sequence analysis. The amino acid sequence of each tryptic peptide of mouse liver mitochondrial medium chain acyl-CoA synthetase differed from that from bovine liver mitochondria only in one or two amino acids. LC-MS/MS analysis provided the information about these differences in amino acid sequences. In addition, we compared the properties of this protein with the homologues from rat and bovine.
中链酰基辅酶A合成酶催化许多外源性羧酸的氨基酸共轭反应的第一步以及脂肪酸代谢。本文报道了对小鼠肝脏中该酶的纯化、特性鉴定及部分氨基酸序列的研究。中链酰基辅酶A合成酶从小鼠肝脏线粒体中分离得到。纯化后的酶不仅能催化直链中链脂肪酸的反应，还能催化芳香酸和芳乙酸的反应。己酸的催化活性最高。在苯环的对位或间位带有甲基、戊基和甲氧基的苯甲酸也能获得较高活性。然而，该酶对丙戊酸和酮洛芬的活性较低。水杨酸无活性。对小鼠和牛肝脏线粒体中的中链酰基辅酶A合成酶进行胶内胰蛋白酶消化，随后进行液相色谱-串联质谱（LC-MS/MS）序列分析。小鼠肝脏线粒体中链酰基辅酶A合成酶的每个胰蛋白酶肽段的氨基酸序列与牛肝脏线粒体的相比，仅在一两个氨基酸上有所不同。LC-MS/MS分析提供了这些氨基酸序列差异的信息。此外，我们还将该蛋白的特性与大鼠和牛的同源蛋白进行了比较。
