Conner M, Krell T, Lindsay J G
Division of Biochemistry and Molecular Biology, University of Glasgow, UK.
Planta. 1996;200(2):195-202. doi: 10.1007/BF00208309.
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 56,000 (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.
在豌豆（Pisum sativum L. cv. Little Marvel）叶片提取物中检测到两种不同的二氢硫辛酰胺脱氢酶（E3s，EC 1.8.1.4），并将其纯化至接近或达到同质状态。主要的酶是一种同型二聚体，其亚基表观分子量（M(r)）值为56,000（占总活性的80 - 90%），通过电喷雾质谱和N端序列分析证实，它与先前研究的线粒体同工型相对应。次要活性（10 - 20%）同样表现为同型二聚体，与叶绿体共纯化，其亚基M(r)值较低，为52,000，这与通过电喷雾质谱测定的52,614 +/- 9.89 Da的M(r)值相近。质体酶在根提取物中的含量也很低，仅占总E3活性的1 - 2%。叶绿体酶的比活性比其线粒体对应物低三到四倍。此外，它对NAD+的亲和力明显更高，对NADH的产物抑制更敏感。它以NADP+为辅因子时无活性，也不受高浓度NADP+或NADPH存在的抑制。针对线粒体酶的抗体与其质体对应物几乎没有交叉反应，现有的氨基酸序列数据也表明这两种酶之间的序列相似性有限。鉴于丙酮酸脱氢酶多酶复合体（PDC）在植物线粒体和叶绿体中的双重定位，不同的叶绿体E3很可能是质体PDC的一个组成部分，因此是迄今为止该复合体中第一个被分离和表征的组分。
