PURIFICATION AND CHARACTERIZATION OF A NOVEL NADPH(NADH)-DEPENDENT HYDROXYPYRUVATE REDUCTASE FROM SPINACH LEAVES - COMPARISON OF IMMUNOLOGICAL PROPERTIES OF LEAF HYDROXYPYRUVATE REDUCTASES
PURIFICATION AND CHARACTERIZATION OF A NOVEL NADPH(NADH)-DEPENDENT HYDROXYPYRUVATE REDUCTASE FROM SPINACH LEAVES - COMPARISON OF IMMUNOLOGICAL PROPERTIES OF LEAF HYDROXYPYRUVATE REDUCTASES
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DOI：
10.1042/bj2500145
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发表时间：
1988-02-15
影响因子：
4.1
通讯作者：
RANDALL, DD
中科院分区：
文献类型：
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作者：
KLECZKOWSKI, LA;RANDALL, DD
从菠菜叶提取物中纯化了一种新的羟丙酮酸还原酶，该酶比NADH更倾向于NADPH作为辅助因子。该酶是一个约70 kDa的低聚物，由两个亚基组成，每个亚基38 kDa。羟丙酮酸（含NADPH）在pH值5.5 ~ 6.5范围内Km约为0.8 mM，在pH值8.2范围内Km约为0.3 mM。pH值为5.5 ~ 6.5时最高，pH值为8.2时下降约65%。pH高于6.0时，羟丙酮酸对底物有较强的抑制作用。还原酶可以使用乙醛酸盐作为替代底物，其速率高达羟丙酮酸盐的四分之一。这种乙醛酸依赖的活性优先于NADPH作为辅助因子。制备的兔抗NADPH(NADH)-羟丙酮酸还原酶抗体对其酶具有高度特异性，并且不与过氧化物酶体NADH（NADPH）依赖性羟丙酮酸还原酶交叉反应，这是通过对菠菜、豌豆和小麦叶提取物的蛋白质进行免疫印迹检测发现的。针对纯化的NADH -羟酸还原酶的抗体也具有高度特异性，仅识别自身抗原。据我们所知，这是文献中首次报道NADPH(NADH)-羟丙酮酸还原酶的发生。由于叶片提取物中新型还原酶的相对较高的比率（至少20 .亩）。Mol /h / mg叶绿素)，该酶可能是乙醇酸途径（光呼吸）的重要侧组分，可能利用从过氧化物酶体“泄漏”的羟丙酮酸，从而促进由乙醇酸衍生的甘油酸池。由于该酶具有乙醛酸依赖活性，因此它也有助于叶片中乙醛酸的形成。
A novel hydroxypyruvate reductase preferring NADPH to NADH as a cofactor was purified over 1500-fold from spinach leaf extracts. The enzyme was an oligomer of about 70 kDa, composed of two subunits of 38 kDa each. The Km for hydroxypyruvate (with NADPH) was about 0.8 mM in the pH range 5.5-6.5, and 0.3 mM at pH 8.2 The Vma. was highest in the pH range 5.5-6.5 and decreased by about 65% at pH 8.2. above pH 6.0, the enzyme was prone to a strong substrate inhibition by hydroxypyruvate. The reductase could use glyoxylate as an alternative substrate, with rates up to one-quarter of those with hydroxypyruvate. This glyoxylate-dependent activity preferred NADPH to NADPH as a cofactor. Rabbit antibodies prepared against NADPH(NADH)-hydroxypyruvate reductase were highly specific for theis enzyme and did not corss-react with peroxisomal NADH(NADPH)-dependent hydroxypyruvate reductase, as found by Western immunoblots of proteins from leaf extracts of spinach, pea and wheat. Antibodies raised against purified NADH(NADPH)-hydeoxyruvate reductase were also highly specific, recognizing only their own antigen. To our knowledge, this is the first report in the literature of the occurrence of NADPH(NADH)-hydroxypyruvate reductases. Because of the relatively high rates of the novel reductase in leaf extracts (at least 20 .mu.mol/h per mg of chlorophyll), this enzyme might be an important side-component of the glycollate pathway (photorespiration), possibly utilizing hydroxypyruvate ''leaked'' from peroxisomes, and thus contributing to the glycerate pool derived from glycollate. Because of the glyoxylate-dependent activity, the enzyme may also contribute to glycollate formation in leaves.