Magnesium-chelatase from developing pea leaves - Characterization of a soluble extract from chloroplasts and resolution into three required protein fractions
Magnesium-chelatase from developing pea leaves - Characterization of a soluble extract from chloroplasts and resolution into three required protein fractions
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DOI：
10.1104/pp.116.2.605
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发表时间：
1998-02-01
期刊：
影响因子：
7.4
通讯作者：
Weinstein, JD
中科院分区：
文献类型：
--
作者：
Guo, RB;Luo, MZ;Weinstein, JD
Mg-螯合酶催化Mg 2+以ATP依赖性插入原卟啉-IX中以形成Mg-原卟啉-IX。这是叶绿素合成的第一步，它位于卟啉利用的分支点;另一个分支导致血红素。以豌豆（Pisum sativum L.）cv Spring）叶绿体，我们已经制备了高活性（1000 pmol 30 min（-1）mg（-1））和稳定形式的Mg-螯合酶。反应有一个滞后的时间过程中，这是克服了与ATP预孵育。ATP和Mg 2+的浓度曲线呈S形，Mg 2+和ATP的表观Km值分别为14.3和0.35 mM。次卟啉的Km为8 nM。该K-m比已发表的卟啉亚铁螯合酶的K-m低300倍。通过蓝色琼脂糖色谱法将可溶性提取物分离成三个级分，然后对柱流出物进行尺寸选择性离心超滤。活性需要所有三个级分，清楚地表明植物Mg-螯合酶需要至少三种蛋白质组分。此外，激活只需要其中两种成分;这两种成分都包含在蓝色琼脂糖柱的流出液中。
Mg-chelatase catalyzes the ATP-dependent insertion of Mg2+ into protoporphyrin-IX to form Mg-protoporphyrin-IX. This is the first step unique to chlorophyll synthesis, and it lies at the branch point for porphyrin utilization; the other branch leads to heme. Using the stromal fraction of pea (Pisum sativum L. cv Spring) chloroplasts, we have prepared Mg-chelatase in a highly active (1000 pmol 30 min(-1) mg(-1)) and stable form. The reaction had a lag in the time course, which was overcome by preincubation with ATP. The concentration curves for ATP and Mg2+ were sigmoidal, with apparent K-m values for Mg2+ and ATP of 14.3 and 0.35 mM, respectively. The K-m for deuteroporphyrin was 8 nM. This K-m is 300 times lower than the published porphyrin K-m for ferrochelatase. The soluble extract was separated into three fractions by chromatography on blue agarose, followed by size-selective centrifugal ultrafiltration of the column flow-through. All three fractions were required for activity, clearly demonstrating that the plant Mg-chelatase requires at least three protein components. Additionally, only two of the components were required for activation; both were contained in the flow-through from the blue-agarose column.