Purification and properties of a factor from rat liver cytosol which stimulates vitamin K epoxide reductase

Two protein type factors which stimulate the reduction of vitamin K₁-2,3-epoxide to vitamin K₁ have been separated from the 105,000g-supernatant fraction (cytosol) of rat liver homogenates. One of these factors is rather labile. However the other factor was sufficiently stable to permit 900-fold purification following sequential column chromatography on DEAE-Sephacel, QAE-Sephadex, CM-Sephadex, and Sephacryl S-200. Four milligrams of this purified material were obtained in 32% yield from 11 g of soluble cytosolic protein. This factor appeared to be homogeneous as determined by gel electrophoresis and has a molecular weight of about 38,000 as determined by gel filtration. The final preparation had no vitamin K epoxide reductase activity in the presence or absence of either NADH or dithiothreitol. The results of kinetic studies using this factor were consistent with its acting as a nonessential activator of the microsome catalyzed reduction of vitamin K₁-2,3-epoxide. The factor did not cause a large change in the apparent K_m (2.2–2.5 μm) of vitamin K epoxide reductase, but the apparent V_max was increased about fourfold.