MAMMALIAN CHOLINEPHOSPHOTRANSFERASE--PURIFICATION & CLONING OF ITS GENE

胆碱磷酸转移酶（CPT）是磷脂酰胆碱（PC）从头合成的终末酶，对肺泡II型细胞PC的酰基化起重要调节作用。这种酶也参与肺巨噬细胞合成血小板聚集因子（1烷基2-乙酰-PC），一种有效的血管抑制剂。尽管一般认为CPT是专门定位于ER膜，我们已经报道，这种酶也存在于外线粒体膜和这两种亚细胞形式的酶之间的性质有一些差异。我们的长期目标是纯化和克隆CPT，并验证CPT的不同同工酶存在的前提。然而，由于与膜结合CPT的溶解而不变性相关的问题的复杂性，开发替代分子生物学策略对于克隆CPT是必不可少的。因此，本项目的主要目标是用根据酵母中已知的CPT基因序列衍生的引物对从豚鼠肝cDNA文库获得的PCR产物进行测序，并使用该序列。以PCR产物为探针，从豚鼠肝cDNA文库中鉴定出CPT的全长cDNA。我们的下一步目标是通过T3聚合酶体外转录CPT cDNA克隆，在兔网织红细胞系统中翻译mRNA，并将35 S-CPT前体多肽导入肺泡II型细胞的线粒体中，以确定CPT在肺泡II型细胞中的线粒体定位。必须指出的是，如果CPT可以显示为如所描述的那样变化并且如所提出的那样定位于线粒体中，这将代表理解线粒体在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我们的努力，从豚鼠肝脏和肺泡II型细胞中溶解和纯化CPT，并验证线粒体和ER中存在CPT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CPT的合理纯化程度。在这种情况下，我们计划开发CPT的纯化方案，最初使用更简单的系统，如肝脏。该系统将允许比较来自两个细胞器的CPT。此外，如所提出的，合理的方法是从肝脏制备CPT并尝试获得特异性抗体。这样的抗体然后可用于制备抗体亲和柱以从肺泡II型细胞分离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.