Siebenlist U, Wohlgemuth S, Finger K, Schweizer E
Lehrstuhl für Biochemie, Universität Erlangen-Nürnberg, Federal Republic of Germany.
Eur J Biochem. 1991 Dec 5;202(2):515-9. doi: 10.1111/j.1432-1033.1991.tb16403.x.
A novel, high-molecular-mass fatty-acid synthetase (FAS) complex has been isolated from streptomycin-bleached Euglena gracilis cells. The enzyme was purified 250-fold from the crude cell homogenate and subsequently migrated upon SDS/PAGE as a single band of molecular mass 270 kDa. This apparent subunit size of the purified protein contrasted with a smaller size of only 200 kDa which was exhibited by the same protein upon immunoblotting of the crude cell extract. The purified Euglena FAS complex cosediments in a sucrose density gradient with yeast FAS and, from this, both enzymes were concluded to have the same overall molecular mass of 2.3 MDa. The enzyme described in this paper appears to be a typical type-I FAS multienzyme which clearly differs from the E. gracilis FAS so far described. Instead, it appears to be organized structurally similar to the type-I FAS multienzymes of lower fungi. In vitro, the purified Euglena FAS complex synthesizes mainly palmitic acid, or its CoA ester, from acetyl CoA and malonyl CoA as substrates. The Km values for acetyl CoA and malonyl CoA are 20 microM and 31 microM, respectively. Similar to the FAS enzymes of other lower eucaryotes, the Euglena type-I FAS is a flavoprotein. In contrast to yeast FAS, however, the flavin cofactor appears to be covalently attached to the enzyme protein. By immunological techniques, the enzyme was shown to be absent in green as well as in etiolated E. gracilis cells, while being rapidly induced upon streptomycin bleaching of heterotrophically growing green cells. The data suggest an inverse correlation between organellar development and derepression of this FAS complex.
从经链霉素处理而变白的纤细裸藻细胞中分离出了一种新型的高分子量脂肪酸合成酶（FAS）复合体。该酶从细胞粗匀浆中纯化了250倍，随后在SDS/PAGE上迁移时呈现为一条分子量为270 kDa的单带。纯化蛋白的这种表观亚基大小与粗细胞提取物免疫印迹时该蛋白呈现的仅200 kDa的较小大小形成对比。纯化的裸藻FAS复合体在蔗糖密度梯度中与酵母FAS共同沉降，由此得出这两种酶的总分子量均为2.3 MDa。本文所述的这种酶似乎是一种典型的I型FAS多酶，明显不同于迄今所描述的纤细裸藻FAS。相反，它在结构上的组织方式似乎与低等真菌的I型FAS多酶相似。在体外，纯化的裸藻FAS复合体主要以乙酰辅酶A和丙二酰辅酶A为底物合成棕榈酸或其辅酶A酯。乙酰辅酶A和丙二酰辅酶A的Km值分别为20 μM和31 μM。与其他低等真核生物的FAS酶相似，裸藻I型FAS是一种黄素蛋白。然而，与酵母FAS不同的是，黄素辅因子似乎与酶蛋白共价连接。通过免疫技术表明，绿色以及黄化的纤细裸藻细胞中均不存在这种酶，而在异养生长的绿色细胞经链霉素处理变白后该酶会迅速被诱导产生。这些数据表明细胞器发育与这种FAS复合体的去阻遏之间存在负相关。
