Wu M, Nie Z Q, Yang J
Department of Biological Sciences, University of Maryland, Baltimore 21228.
Plant Cell. 1989 May;1(5):551-7. doi: 10.1105/tpc.1.5.551.
From a high-salt extract of the purified thylakoid membrane, an 18-kD protein was detected. This protein was translated by the chloroplast ribosomes and could form a stable DNA-protein complex with a cloned chloroplast DNA replicative origin [Nie, Z.Q., Chang, D.Y., and Wu, M. (1987) Mol. Gen. Genet. 209, 265-269]. In this paper, the 18-kD protein is linked to frxB, a chloroplast-encoded, ferredoxin-type, iron-sulfur protein, by N-terminal microsequencing of the purified protein and computer analysis. The identification is further supported empirically by the fact that the electron paramagnetic resonance spectra of the protein indicate the presence of iron-sulfur clusters. A polyclonal antibody raised against a synthetic pentadecameric peptide with amino acid sequence corresponds to the highly conserved region of the frxB protein and reacts strongly and specifically with the 18-kD protein band in protein gel blot analyses. The 18-kD iron-sulfur protein is found to be related to a subunit of the respiratory chain NADH dehydrogenase by its cross-reaction with a polyclonal antibody raised against highly purified NADH-ubiquinone oxidoreductase, a key enzyme of the respiratory chain. These data are consistent with chlororespiration, and, thus, possible implication of chlororespiration in regulating the initiation of chloroplast DNA replication is discussed.
从纯化的类囊体膜的高盐提取物中检测到一种18-kD蛋白。该蛋白由叶绿体核糖体翻译，并能与克隆的叶绿体DNA复制起点形成稳定的DNA-蛋白复合物[Nie, Z.Q., Chang, D.Y., and Wu, M. (1987) Mol. Gen. Genet. 209, 265 - 269]。在本文中，通过对纯化蛋白的N端微量测序和计算机分析，将18-kD蛋白与frxB（一种叶绿体编码的铁氧化还原蛋白型铁硫蛋白）联系起来。该蛋白的电子顺磁共振光谱表明存在铁硫簇，这一事实从经验上进一步支持了这一鉴定。用一种具有氨基酸序列的合成十五聚体肽制备的多克隆抗体，该序列对应于frxB蛋白的高度保守区域，并且在蛋白质凝胶印迹分析中与18-kD蛋白条带发生强烈且特异性的反应。通过与针对高度纯化的NADH-泛醌氧化还原酶（呼吸链的关键酶）制备的多克隆抗体发生交叉反应，发现18-kD铁硫蛋白与呼吸链NADH脱氢酶的一个亚基有关。这些数据与叶绿体呼吸作用一致，因此讨论了叶绿体呼吸作用在调节叶绿体DNA复制起始方面可能的作用。
