The goal of this project is to understand the mechanisms which regulate CTP: phosphorylcholine Cytidylyltransferase activity in type II pneumonocytes isolated from adult and fetal lung and to relate these mechanisms to the control of the synthesis of phosphatidylcholine and dipalmitoylphosphatidylcholine (DPPC) for pulmonary surfactant formation. Since defects in the pulmonary surfactant system are associated with several pulmonary diseases, a detailed understanding of the control of surfactant formation is of major importance. Isolated type II cells from adult and fetal rat lung will be used.
The specific aims are: 1) to characterize the molecular forms of cytidylyltransferase in type II cells, 2) to examine the fatty acid - dependent translocations of cytidylyltransferase, 3) to assess the significance of phosphorylation of cytidylyltransferase as a regulatory process, 4) to determine the effects of dexamethasone, thyroxine and fibroblast pneumocyte factor on the intracellular forms of cytidylyltransferase and 5) to relate the regulatory properties of cytidylyltransferase with changes in phosphatidylcholine and DPPC synthesis. The study applies the already developed methods for the purification of cytidylyltransferase to prepare purified enzyme and to raise polyclonal antibodies. The antibodies will in turn be used to isolate and characterize cytidylyltransferase from type II cells and to establish immunochemical methods to measure cytidylyltransferase. Cells from fetal lung will be isolated and cultured in the presence of carbon-stripped bovine fetal calf serum and induced to develop type II cell characteristics by hormonal treatment. The subunit composition, cytosolic forms and membrane-bound forms will be determined by SDS-PAGE and immunoblotting techniques. Translocation will be measured both by activity measurements and by immunochemical detection. Phosphorylation and biosynthesis of cytidylyltransferase will be determined by 32Pi and [35S] methionine incorporation by culture cells followed by immunoprecipitation of cytidylyltransferase.
