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.
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