MAMMALIAN CHOLINEPHOSPHOTRANSFERASE--PURIFICATION & CLONING OF ITS GENE

胆碱磷酸转移酶(CPT)是磷脂酰胆碱(PC)从头合成的末端酶，在调节肺泡II型细胞PC的酰基方面起着重要作用。该酶还参与肺巨噬细胞合成血小板聚集因子(1-烷基-2-乙酰基-PC)，这是一种有效的血管抑制因子。尽管普遍认为CPT仅定位于内质网细胞膜，但我们已报道该酶也存在于线粒体外膜，并且这两种亚细胞形式的酶在性质上存在一些差异。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，由于膜结合型CPT在无变性的情况下增溶问题的复杂性，开发一种替代的分子生物学策略对于克隆CPT是至关重要的。因此，本课题的主要目的是根据已知的酵母菌CPT基因序列设计引物，对从豚鼠肝脏cDNA文库中获得的扩增产物进行测序，并利用该序列进行序列分析。验证了PCR产物作为探针可用于从豚鼠肝脏cDNA文库中鉴定CPT的全长cDNA。我们的下一个目标是用T3聚合酶体外转录CPT基因克隆，在兔网织红细胞系统中翻译mRNA，并将35S-CPT前体多肽导入到从肺泡II型细胞分离的线粒体中，以证实CPT在肺泡II型细胞中的线粒体定位。必须指出的是，如果CPT能够被证明如所描述的那样变化并且如所提议的那样定位于线粒体，这将代表着在理解线粒体在POC生物合成中的作用方面的重要进展。本项目的第三个目标是继续从豚鼠肝脏和肺泡II型细胞中分离和纯化CPT，并验证CPT在线粒体和内质网中存在不同同工酶的假设。重要的是要指出，在任何系统中都从未实现过合理程度的CPT纯化。在这种情况下，我们计划首先用一个更简单的系统，如肝脏，来开发CPT的纯化方案。该系统将允许比较两个细胞器的CPT。此外，如建议的那样，一个合理的方法是从肝脏制备CPT，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将CPT从肺泡型II型细胞中分离出来。

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.