MAMMALIAN CHOLINEPHOSPHOTRANSFERASE--PURIFICATION & CLONING OF ITS GENE

哺乳动物胆碱磷酸转移酶--纯化

• 批准号: 6485269
• 负责人: SALIL K DAS
• 金额: $ 17.91万
• 依托单位: MEHARRY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6485269
• 关键词: choline; enzyme mechanism; genetic library; guinea pigs; isozymes; laboratory mouse; laboratory rabbit; membrane proteins; molecular cloning; other phosphotransferase; protein localization; protein purification

Cholinephosphotransferase (CPT), the terminal enzyme in the de novo synthesis of phosphatidylcholine (PC), plays an important role in regulating the acyl group of PC in alveolar type II cells. This enzyme is also involved in lung macrophages in the synthesis of platelet aggregation factor (1 alkyl 2-acetyl-PC), a potent vasodepressor. In spite of the general belief that CPT is exclusively localized in the ER membrane, we have reported that this enzyme is also present in the outer mitochondrial membrane and there is some difference in the properties between these two subcellular forms of the enzyme. Our long term goal is to purify and clone CPT and validate the premise that different isozymes of CPT are present. However, because of the complexity of the problems associated with the solubilization of the membrane-bound CPT without denaturation, development of an alternative molecular biology strategy is essential to clone CPT. Therefore, the primary goal of this project is to sequence the PCR products obtained from guinea pig liver cDNA library with primers derived on the basis of the known CPT gene sequence in yeast, and to use the sequence. Verified PCR product as a probe to identify a full length cDNA for CPT from guinea pig liver cDNA library. Our next goal is to confirm mitochondrial localization of CPT in alveolar type II cell by transcribing CPT cDNA clone in vitro with T3 polymerase, translating the mRNA in a rabbit reticulocyte system and importing 35S- CPT precursor polypeptides into mitochondria isolated from alveolar type II cells. It must be noted that if the CPT can be shown to vary as described and to be localized in mitochondria as proposed, this would represent an important advancement in understanding the role of mitochondria in POC biosynthesis. The third objective of this project is to continue our efforts to solubilize and purify CPT from guinea pig liver and alveolar type II cells and validate the premise that different isozymes of CPT are present in both mitochondria and ER. It is important to point out that a reasonable degree of purification of CPT has never been achieved in any system. This being the case, we plan to develop the purification scheme for the CPT initially with a simpler system, such the liver. This system would permit comparing CPT from the two organelles. Further, a reasonable approach, as proposed, is to prepare CPT from liver and try to obtain specific antibodies. Such antibodies may then be useful to prepare an antibody affinity column to isolate CPT from alveolar type II cells.

胆碱磷酸转移酶（CPT），磷脂酰胆碱（PC）的从头合成中的末端酶（PC）在调节肺泡II型细胞中PC的酰基PC群中起重要作用。该酶还参与了肺巨噬细胞的血小板聚集因子（1烷基2-乙酰-PC）的合成，这是一种有效的加速压迫剂。尽管人们普遍认为CPT仅位于ER膜中，但我们报告说，该酶也存在于线粒体外膜中，并且这两种亚细胞形式的酶的性质存在差异。我们的长期目标是纯化和克隆CPT并验证存在CPT的不同同工酶的前提。但是，由于与膜结合的CPT溶解相关的问题的复杂性而无需变性，因此开发替代分子生物学策略对于克隆CPT至关重要。因此，该项目的主要目标是对从豚鼠肝cDNA文库获得的PCR产物进行测序，其引物根据酵母中已知的CPT基因序列得出，并使用该序列。验证的PCR产物是从豚鼠肝cDNA文库中识别CPT的全长cDNA的探针。我们的下一个目标是通过将CPT cDNA克隆在体外用T3聚合酶转录CPT在肺泡II型细胞中的线粒体定位，并将MRNA转化为兔网状细胞系统中的mRNA，并将35S-CPT-CPT前体procursor多肽转化为与肺泡型II细胞分离的线粒体。必须指出的是，如果可以表明CPT如所述变化并按照提出的线粒体定位，则这将代表理解线粒体在POC生物合成中的作用方面的重要进步。该项目的第三个目标是继续我们从豚鼠肝脏和牙槽II型细胞中溶解和纯化CPT的努力，并验证线粒体和ER中存在不同同工酶存在不同同工酶的前提。重要的是要指出，在任何系统中从未实现合理的CPT纯化。在这种情况下，我们计划使用更简单的系统（例如肝脏）开发CPT的纯化方案。该系统将允许比较两个细胞器的CPT。此外，提出的合理方法是从肝脏中制备CPT并尝试获得特定的抗体。然后，此类抗体可能有助于制备抗体亲和力柱以分离牙槽II型细胞。