The gamma-glutamyl carboxylase makes an essential modification in the vitamin K-dependent blood coagulation factors, two known bone proteins, and probably, additional yet unidentified proteins. The carboxylase is also an interesting protein because of the multiple co-factors (oxygen and reduced vitamin K) involved in its function. The carboxylase has obvious implications for health; lack of the carboxylase results in bleeding disorders and congenital bone deformities. Our long-term goal is to understand what structural features are important for its various functions. In this project our specific research objectives are to: 1. Finish the cDNA sequence of both human and bovine carboxylase cDNA molecules and prove that the gamma-glutamyl carboxylase has been isolated by: a. expressing the cDNA in a human cell line that is known to express undercarboxylated factor IX and to demonstrate increased levels of carboxylation; b. expressing the carboxylase in E. coli as a fusion protein, making antibodies and attempting to immunoprecipitate the carboxylase activity; c. using anti-sense oligonucleotides to attempt to arrest transcription or translation of the carboxylase. 2. Prepare large amounts of the carboxylase for additional characterizations. 3. Determine the relative amount of carboxylase produced in different tissues, the chromosomal location of the gene for the carboxylase, and the sequence of the intron-exon borders of the genomic sequence of the carboxylase gene. 4. Determine if there are two functional domains of the carboxylase enzyme and define the portion(s) of the carboxylase molecule responsible for its interaction with the propeptide of the vitamin K-dependent proteins. Make mutations suggested by the above experiments. 5. Examine some aspects of the mechanism of the carboxylation reaction.