Identification and purification of a distinct dihydrolipoamide dehydrogenase from pea chloroplasts
Identification and purification of a distinct dihydrolipoamide dehydrogenase from pea chloroplasts
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DOI：
10.1007/bf00208309
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发表时间：
1996-10-01
期刊：
影响因子：
4.3
通讯作者：
Lindsay, JG
中科院分区：
文献类型：
--
作者：
Conner, M;Krell, T;Lindsay, JG
在豌豆（Pisum sativum L.）中检测到两种不同的二氢硫辛酰胺脱氢酶（E3 s，EC 1.8.1.4）。CV. Little Marvel）叶提取物并纯化至均匀或接近均匀。主要酶是一种同二聚体，表观亚基M（r）值为56000（占总活性的80-90%），与之前研究的线粒体亚型相对应，这一点通过电喷雾质谱和N末端序列分析得到证实。较小的活性（10-20%）也表现为同二聚体，与叶绿体共纯化，并显示较低的亚基M（r）值52 000，其接近电喷雾质谱测定的M（r）值52 614 +/- 9.89 Da。质体酶在根提取物中也以低水平存在，仅占总E3活性的1-2%。叶绿体酶的比活性比线粒体酶低三到四倍。此外，它对NAD（+）的亲和力明显更高，对NADH的产物抑制更敏感。当NADP（+）作为辅因子时，它不表现出活性，也不受高浓度NADP（+）或NADPH的抑制。线粒体酶的抗体显示很少或没有交叉反应性与其质体对应物和可用的氨基酸序列数据也表明只有有限的序列相似性之间的两种酶。鉴于丙酮酸脱氢酶多酶复合物（PDC）在植物线粒体和叶绿体中的双重位置，很可能不同的叶绿体E3是质体PDC的组成部分，因此代表了迄今为止分离和表征的该复合物的第一组分。
Two distinct dihydrolipoamide dehydrogenases (E3s, EC 1.8.1.4) have been detected in pea (Pisum sativum L. cv. Little Marvel) leaf extracts and purified to at or near homogeneity. The major enzyme, a homodimer with an apparent subunit M(r) value 56000 (80-90% of overall activity), corresponded to the mitochondrial isoform studied previously, as confirmed by electrospray mass spectrometry and N-terminal sequence analysis. The minor activity (10-20%), which also behaved as a homodimer, copurified with chloroplasts, and displayed a lower subunit M(r) value of 52 000 which was close to the M(r) value of 52 614 +/- 9.89 Da determined by electrospray mass spectrometry. The plastidic enzyme was also present at low levels in root extracts where it represented only 1-2% of total E3 activity. The specific activity of the chloroplast enzyme was three- to fourfold lower than its mitochondrial counterpart. In addition, it displayed a markedly higher affinity for NAD(+) and was more sensitive to product inhibition by NADH. It exhibited no activity with NADP(+) as cofactor nor was it inhibited by the presence of high concentrations of NADP(+) or NADPH. Antibodies to the mitochondrial enzyme displayed little or no cross-reactivity with its plastidic counterpart and available amino acid sequence data were also suggestive of only limited sequence similarity between the two enzymes. In view of the dual location of the pyruvate dehydrogenase multienzyme complex (PDC) in plant mitochondria and chloroplasts, it is likely that the distinct chloroplastic E3 is an integral component of plastidic PDC, thus representing the first component of this complex to be isolated and characterised to date.