MAMMALIAN CHOLINEPHOSPHOTRANSFERASE--PURIFICATION & CLONING OF ITS GENE

胆碱磷酸酯转移酶（Cholinephosphotransferase， CPT）是肺泡II型细胞从头合成磷脂酰胆碱（phosphatidylcholine， PC）的末端酶，在调节磷脂酰胆碱（PC）的酰基中起重要作用。该酶还参与肺巨噬细胞血小板聚集因子（1烷基2乙酰- pc）的合成，这是一种有效的血管抑制剂。尽管人们普遍认为CPT仅局限于内质网膜，但我们已经报道了这种酶也存在于线粒体外膜，并且在这两种亚细胞形式的酶之间存在一些特性差异。我们的长期目标是纯化和克隆CPT，并验证CPT存在不同同工酶的前提。然而，由于膜结合CPT不变性的增溶问题的复杂性，开发一种替代的分子生物学策略对于克隆CPT至关重要。因此，本项目的主要目标是利用已知酵母CPT基因序列衍生的引物，对从豚鼠肝脏cDNA文库中获得的PCR产物进行测序，并使用该序列。验证PCR产物作为探针从豚鼠肝脏cDNA文库中鉴定CPT全长cDNA。我们的下一个目标是通过在体外用T3聚合酶转录CPT cDNA克隆，在兔网织细胞系统中翻译mRNA，并将35S- CPT前体多肽导入肺泡II型细胞分离的线粒体中，来确认CPT在肺泡II型细胞中的线粒体定位。必须指出的是，如果CPT可以像所描述的那样变化，并且像所提出的那样定位在线粒体中，这将代表着理解线粒体在POC生物合成中的作用的重要进展。本项目的第三个目标是继续努力从豚鼠肝脏和肺泡II型细胞中溶解和纯化CPT，并验证CPT在线粒体和内质网中都存在不同同工酶的前提。重要的是要指出，在任何系统中，从未实现过合理程度的CPT净化。在这种情况下，我们计划首先用一个更简单的系统（如肝脏）开发CPT的净化方案。该系统将允许比较两种细胞器的CPT。此外，一个合理的方法，如所建议的，是制备CPT从肝脏，并试图获得特异性抗体。这样的抗体可用于制备抗体亲和柱以从肺泡II型细胞中分离CPT。

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.