The long-term objective of this proposal is to understand vitamin K-dependent biosynthesis of proteins and how these processes are regulated. We will focus on this reaction in liver and isolated cells from lung, epithelium and human carcinomas. Special emphasis is placed on the biosynthesis of blood coagulation factors. A major part of the proposed work focusses on an endonuclease eta (endo eta) insensitive protein that constitutes the major gamma- carboxylated product of the vitamin K-dependent carboxylation reaction in vitro. Preliminary experiments suggest that the protein is a component of the vitamin K-dependent carboxylase enzyme complex and we propose that the protein is a CO2-carrier in the gamma-carboxylation reaction. Experiments are designed to test this hypothesis. Another part of this proposal focusses on gamma-carboxylation of protein precursors of coagulation factor II in rat and human liver. We would like to understand why selected precursors only serve as carboxylase substrates in vitro. The experimental approach is based on a systematic comparison of two precursor forms of clotting factor II and uses peptide mapping of 125I-iodinated proteins and mapping by HPLC. Mononuclear phagocytes are potent triggers of the coagulation system. Pulmonary macrophages produce tissue factor and factor V but appear to be devoid of carboxylase activity. On the other hand, Type II pneumocytes exhibit significant carboxylase activity. Experiments are proposed to test whether or not the lung can provide all the necessary coagulation factors for extrinsic coagulation in the lung. Macrophages are known to stimulate proliferation of Type II cells. Therefore, Type II cells will be cultured in conditioned medium from stimulated macrophages to test the possibility of communication between macrophages and type II cells which can regulate procoagulant output by Type II cells. Antibodies against rat prothrombin and factor X will be used to identify 14CO2-Labeled protein precursors in an in vitro carboxylation system of Type II cells. The same approach will be used to study biosynthesis of vitamin K-dependent proteins by human endothelial cells. Identification of vitamin K-dependent proteins produced by various human carcinoma cell lines will also be attempted. Finally, purification of the physiologically important vitamin K1 reducing dehydrogenase enzyme in liver microsomes that mediates the antidotic effect of vitamin K1 will be attempted after proteolytic cleavage of the microsomal membrane with protease.
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