菠菜叶片β-酮酰-[酰基载体蛋白]合酶的分离与功能。
Isolation and function of spinach leaf beta-ketoacyl-[acyl-carrier-protein] synthases.
机构信息
Department of Biochemistry and Biophysics, University of California, Davis, California 95616.
出版信息
Proc Natl Acad Sci U S A. 1982 Oct;79(19):5808-12. doi: 10.1073/pnas.79.19.5808.
Crude spinach leaf extract readily forms the stearoyl derivative of acyl-carrier-protein (ACP) when acetyl-ACP and malonyl-ACP are incubated together. Palmitoyl-ACP is also elongated by malonyl-ACP to stearoyl-ACP. When beta-ketoacyl-ACP synthase {3-oxoacyl-[ACP] synthase; acyl-[ACP]:malonyl-[ACP] C-acyltransferase (decarboxylating), EC 2.3.1.41} is purified with decanoyl-ACP as the assay substrate, palmitoyl-ACP elongation activity is lost. When palmitoyl-ACP is the assay substrate, another protein is isolated that specifically elongates palmitoyl-ACP to beta-ketostearoyl-ACP but has no activity towards decanoyl-ACP. The first protein is designated beta-ketoacyl-ACP synthase I and participates in the conversion of acetyl-ACP to palmitoyl-ACP, whereas the second protein is designated beta-ketoacyl-ACP synthase II, and its substrate specificity is highly restricted to myristoyl-ACP and palmitoyl-ACP. The purification of synthase II is described, and its activity is compared to synthase I. Reconstitution experiments with the highly purified nonassociated enzymes in fatty acid synthesis plus synthases I and II clearly demonstrate the roles of these two proteins in fatty acid synthesis.
粗提的菠菜叶提取物在乙酰-ACP 和丙二酰-ACP 共同孵育时，很容易形成酰基载体蛋白（ACP）的硬脂酰衍生物。棕榈酰-ACP 也可以被丙二酰-ACP 延长为硬脂酰-ACP。当β-酮酰-ACP 合酶{3-氧酰基-[ACP]合酶；酰基-[ACP]：丙二酰-[ACP] C-酰基转移酶（脱羧），EC 2.3.1.41}用癸酰-ACP 作为测定底物进行纯化时，棕榈酰-ACP 延长活性丧失。当棕榈酰-ACP 是测定底物时，分离出另一种蛋白质，它特异性地将棕榈酰-ACP 延长为β-酮硬脂酰-ACP，但对癸酰-ACP 没有活性。第一种蛋白质被指定为β-酮酰-ACP 合酶 I，参与乙酰-ACP 向棕榈酰-ACP 的转化，而第二种蛋白质被指定为β-酮酰-ACP 合酶 II，其底物特异性高度局限于肉豆蔻酰-ACP 和棕榈酰-ACP。描述了合酶 II 的纯化，并比较了其与合酶 I 的活性。在脂肪酸合成中使用高度纯化的非相关酶以及合酶 I 和 II 的重组实验清楚地表明了这两种蛋白质在脂肪酸合成中的作用。
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