Sonnenborn U, Kunau W H
Biochim Biophys Acta. 1982 Sep 14;712(3):523-34. doi: 10.1016/0005-2760(82)90280-6.
De novo biosynthesis of fatty acids in the heterotrophic marine dinoflagellate, Crypthecodinium cohnii, has been studied in vitro. Fatty acid synthetase was located in the cytosol and its activity was dependent on acetyl-CoA, malonyl-CoA, NADPH2 and NADH2. The enzyme was purified 100-fold using ion-exchange chromatography on DEAE-Sephadex A-25, adsorption to hydroxyapatite and gel filtration on Sepharose 4B columns. Very active endogenous proteases were separated from the fatty acid synthetase at the first step of purification. The purified enzyme had a molecular weight of about 400000, as judged from gel filtration, sucrose density gradient centrifugation and polyacrylamide gel electrophoresis under non-denaturing conditions. Polyacrylamide gel electrophoresis under denaturing conditions in the presence of SDS and urea revealed one major protein band of Mr 180000, suggesting that the enzyme is composed of two multifunctional subunits of apparently identical molecular weight. Reaction products of the C. cohnii fatty acid synthetase are free fatty acids due to the presence of a thioesterase activity in the purified enzyme complex. The main product is palmitate. Docosahexaenoic acid (C22:6, n-3), the major fatty acid component of C. cohnii lipids, is not directly synthesized by the enzyme.
已对异养海洋甲藻寇氏隐甲藻（Crypthecodinium cohnii）中脂肪酸的从头生物合成进行了体外研究。脂肪酸合成酶位于细胞质中，其活性依赖于乙酰辅酶A、丙二酰辅酶A、还原型辅酶Ⅱ（NADPH2）和还原型辅酶Ⅰ（NADH2）。通过在二乙氨基乙基葡聚糖A-25（DEAE-Sephadex A-25）上进行离子交换色谱、吸附到羟基磷灰石以及在琼脂糖4B柱上进行凝胶过滤，该酶被纯化了100倍。在纯化的第一步，非常活跃的内源性蛋白酶与脂肪酸合成酶分离。从凝胶过滤、蔗糖密度梯度离心以及非变性条件下的聚丙烯酰胺凝胶电泳判断，纯化后的酶分子量约为400000。在十二烷基硫酸钠（SDS）和尿素存在的变性条件下进行聚丙烯酰胺凝胶电泳，显示出一条主要的分子量为180000的蛋白带，这表明该酶由两个分子量明显相同的多功能亚基组成。由于纯化的酶复合物中存在硫酯酶活性，寇氏隐甲藻脂肪酸合成酶的反应产物是游离脂肪酸。主要产物是棕榈酸。二十二碳六烯酸（C22:6，n-3）是寇氏隐甲藻脂质的主要脂肪酸成分，不是由该酶直接合成的。
