Vitamin K is now known to catalyze the post translational carboxylation of ten residues of glutamic acid in a prothrombin precursor protein in the endoplasmic reticulum of mammalian liver to form Gamma-carboxyglutamic acid (Gla) residues. The enzyme system responsible for this vitamin K-dependent carboxylation is membrane-bound and can be solubilized with a number of detergents. The precise nature ofthe biochemical mechanism by which carbon dioxide is introduced into peptide-bound glutamate is still unknown.
The specific aims of this research project are: 1) To purify the membrane-bound vitamin K-dependent Glu-peptide carboxylase to homogeneity: 2) To determine the mechanism by which vitamin K catalyzes the carboxylation of Glu-peptides to form Gla-peptides and the action of the coumarin drugs in inhibiting the process. Chemical models, as well as biochemical systems will be studied; 3) To study the importance of the vitamin K cycle in normal and warfarin-resistant rats in vivo; 4) To reassemble the vitamin K carboxylase system in artificial membrane from purified components. In order to achieve these goals, modern technology designed to purify membrane-bound proteins and to detect, isolate, and determine the structures of vitamin K intermediates will be employed. These include high-pressure liquid chromatography, gas-liquid chromatography coupled to mass spectrometry, and electron-spin resonance spectrometry.
