PROPERTIES OF A CHLOROPLAST ENZYME THAT CLEAVES THE CHLOROPHYLL A/B BINDING-PROTEIN PRECURSOR - OPTIMIZATION OF AN ORGANELLE-FREE REACTION
PROPERTIES OF A CHLOROPLAST ENZYME THAT CLEAVES THE CHLOROPHYLL A/B BINDING-PROTEIN PRECURSOR - OPTIMIZATION OF AN ORGANELLE-FREE REACTION
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DOI：
10.1104/pp.90.1.117
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发表时间：
1989-05-01
期刊：
影响因子：
7.4
通讯作者：
LAMPPA, GK
中科院分区：
文献类型：
--
作者：
ABAD, MS;CLARK, SE;LAMPPA, GK
高等植物 [Pisum sativum] 叶绿体的主要捕光叶绿素 a/b 结合蛋白 (LHCP) 是核编码的，作为前体合成，并在进口时进行加工。我们之前（GK Lamppa，M. Abad [1987] J. Cell Biol. 105：2641-2648）鉴定了一种可裂解 LHCP 前体（pLHCP）的可溶性酶。在这项研究中，我们描述了加工活性最佳恢复的条件，并提供了 pLHCP N 末端确实被切割、去除转运肽的证据。从克隆的 pLHCP 基因中进行了两次 pLHCP 删除，分别从蛋白质的羧基末端去除了 13 个和 21 个氨基酸。经过无细胞器处理后，裂解产物在 SDS-PAGE 过程中显示出与前体截断大小成比例的迁移率变化，正如 N 端处理所预测的那样。出乎意料的是，尽管转运肽结构域完整，但缺少 C 末端 91 个残基的第三个截短前体并未被切割，表明这种缺失破坏了加工所需的前体的构象特征。 pLHCP 加工酶可被 2 毫摩尔 EDTA 和 0.4 毫摩尔金属螯合剂 1,10 菲咯啉抑制，而仅在高浓度时被 EGTA 抑制，并且对碘乙酸不敏感。最佳处理温度为 pH 8 至 9，温度为 26°C。 C.凝胶过滤色谱显示pLHCP加工酶的表观分子量约为240,000。处理 pLHCP 的相同柱级分还将核酮糖-1,5-二磷酸羧化酶小亚基的前体转化为其成熟形式。
The major light-harvesting chlorophyll a/b binding protein (LHCP) of higher plant [Pisum sativum] chloroplasts is nuclear-encoded, synthesized as a precursor, and processed upon import. We have previously (GK Lamppa, M. Abad [1987] J. Cell Biol. 105: 2641-2648) identified a soluble enzyme that cleaves the LHCP precursor (pLHCP). In this study, we describe the conditions for optimal recovery of the processing activity and provide evidence that the N terminus of pLHCP is indeed cleaved, removing the transit peptide. Two pLHCP deletions were made from a cloned pLHCP gene removing 13 and 21 amino acids, respectively, from the carboxy terminus of the protein. After organelle-free processing, the cleavage products showed a shift in mobility during SDS-PAGE proportional to the size of the precursor truncations, as predicted for N-terminal processing. Unexpectedly, a third truncated precursor lacking 91 residues of the C-terminus was not cleaved although the transit peptide domain was intact, suggesting that this deletion disrupted conformational features of the precursor necessary for processing. The pLHCP processing enzyme is inhibited by 2 millimolar EDTA and the metal chelator 1,10 phenanthroline at 0.4 millimolar, while being inhibited by EGTA only at high concentrations and insensitive to iodoacetate. Optimal processing occurs at pH 8 to 9, and 26.degree. C. Gel filtration chromatography shows that the pLHCP processing enzyme has an apparent molecular weight of about 240,000. The identical column fractions that process pLHCP also convert the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase to its mature form.