Shorrosh B S, Savage L J, Soll J, Ohlrogge J B
Department of Botany and Plant Pathology, Michigan State University, East Lansing 48824-1321, USA.
Plant J. 1996 Aug;10(2):261-8. doi: 10.1046/j.1365-313x.1996.10020261.x.
Two forms of acetyl-CoA carboxylase (ACCase) have been characterized in pea (Pisum sativum L.) leaves; a heteromeric chloroplast enzyme and a homomeric, presumably cytosolic enzyme. The biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and beta-carboxyltransferase (CT) subunits of the plastidial-ACCase have recently been characterized and cloned. To further characterize the carboxyltransferase, an improved assay for CT was developed and used to follow its partial purification. CT activity co-purifies with ACCase activity during gel permeation chromatography. However, upon anion-exchange chromatography or native PAGE, CT separates from the BC and BCCP subunits of plastidial-ACCase and ACCase activity is lost. In addition, it is demonstrated that a previously sequenced pea chloroplast cDNA of unknown function (IEP96) with a predicted molecular weight of 91 kDa encodes the alpha-CT subunit of the MS-ACCase. Antibodies raised against the first 404 amino acids of IEP96 protein detected a polypeptide with molecular weight of 91 kDa that co-eluted during gel permeation chromatography with plastidial CT and ACCase activities. These antibodies also immunoprecipitated the activities of both ACCase and CT with the concomitant precipitation of the beta-CT subunit. Furthermore, antibodies against beta-CT immunoprecipitated the IEP96 protein. Two-dimensional PAGE and DEAE purification of ACCase protein demonstrated that the beta-CT forms a tight association with the IEP96 protein. Pea leaf was fractionated into soluble and membrane fractions and the alpha-CT subunit was primarily associated with the membrane fraction. Together, these data demonstrate that IEP96 is the alpha-CT subunit of pea chloroplast ACCase.
在豌豆（Pisum sativum L.）叶片中已鉴定出两种形式的乙酰辅酶A羧化酶（ACCase）；一种是异源多聚体叶绿体酶，另一种是同源多聚体，可能是胞质酶。质体ACCase的生物素羧化酶（BC）、生物素羧基载体蛋白（BCCP）和β-羧基转移酶（CT）亚基最近已被鉴定和克隆。为了进一步鉴定羧基转移酶，开发了一种改进的CT测定方法，并用于跟踪其部分纯化过程。在凝胶渗透色谱过程中，CT活性与ACCase活性共纯化。然而，在阴离子交换色谱或非变性聚丙烯酰胺凝胶电泳时，CT与质体ACCase的BC和BCCP亚基分离，ACCase活性丧失。此外，已证明一个先前测序的功能未知的豌豆叶绿体cDNA（IEP96），预测分子量为91 kDa，编码MS-ACCase的α-CT亚基。针对IEP96蛋白前404个氨基酸产生的抗体检测到一种分子量为91 kDa的多肽，该多肽在凝胶渗透色谱过程中与质体CT和ACCase活性共洗脱。这些抗体还免疫沉淀了ACCase和CT的活性，并伴随β-CT亚基的沉淀。此外，针对β-CT的抗体免疫沉淀了IEP96蛋白。ACCase蛋白的二维聚丙烯酰胺凝胶电泳和二乙氨基乙基纤维素纯化表明，β-CT与IEP96蛋白紧密结合。将豌豆叶片分离为可溶性部分和膜部分，α-CT亚基主要与膜部分相关。总之，这些数据表明IEP96是豌豆叶绿体ACCase的α-CT亚基。
