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 Km (2.2-2.5 microM) of vitamin K epoxide reductase, but the apparent Vmax was increased about fourfold.

Vitamin K requirement and the concentration of vitamin K in rat liver.

The concentration of vitamin K was determined in the liver of different strains of rats, and in male and female warfarin-resistant rats by feeding 3H-vitamin K in a purified diet. In each case, the level of vitamin K in the liver correlated approximately with the amount of vitamin K fed. The results indicate that differences in the requirement for vitamin K between the sexes and between strains of rats are due principally to different required concentrations of vitamin K in liver and not to differences in absorption or turnover of the vitamin. The results of the determination of vitamin K epoxide levels in male and female warfarin-resistant rats, and other data, suggest that the amount of vitamin K required in liver may be in part due to differences in the activity of the enzyme, vitamin K epoxide reductase.

Bile acids. XLIV, quantitation of bile acids from the bile fistula rat given (4-14C) cholesterol.

The bile acids derived from [4-14-C]cholesterol administered intracardially to rats with cannulated bile ducts were identified and quantitated. Over a period of 28 days about 90% of the administered 14-C was found in bile of which 73% was retained in the biliary acid fraction. [7beta-3-H]cholic acid, alpha-muri[3beta-3-H]cholic acid, beta-muri[3beta-3-H]cholic acid and litho[3beta-3-H]cholic acid were prepared with specific activities of about 30 muCi/mg by reduction of appropriate ketonic precursors with NaB3H4 and were added to the biliary acid fraction. After separation and purification of the bile acids, cholic, chenodeoxycholic, alpha- and beta-muricholic acids accounted for 70, 16, 7.5 and 6.1%, respectively, of the 14-C in the biliary acid fraction. The specific activities of these isolated 14-C-labeled acids were almost identical. Lithocholic acid accounted for a maximum of 0.2% and ursodeoxycholic acid and 7-oxolithocholic acid could account for no more than 2% of the biliary 14-C. Gas-liquid chromatography on 3% OV-17 of the trimethylsilyl ether derivatives of the methyl esters of the common bile acids of rat bile results in their complete separation and provides a convenient means of estimating the relative proportions of these acids in rat bile. By this method, the relative amounts of the four major acids, cholic, chenodeoxycholic, alpha- and beta-muricholic acids were 63, 20, 8 and 6%, respectively.

Separation of bile acids of rat bile by thin-layer chromatography.

The common bile acids of rat bile (chenodeoxycholic, hyodeoxycholic, cholic, alpha-muricholic, and beta-muricholic acids) are completely separated by a new thin-layer chromatographic system.