RESUMO
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.
Assuntos
Fígado/enzimologia , Oxigenases de Função Mista/metabolismo , Animais , Citosol/enzimologia , Ativação Enzimática , Concentração de Íons de Hidrogênio , Cinética , Masculino , Peso Molecular , Ratos , Vitamina K Epóxido RedutasesAssuntos
Transtornos da Coagulação Sanguínea/sangue , Protrombina/metabolismo , Vitamina K/fisiologia , Ácido 1-Carboxiglutâmico/metabolismo , Aminoácidos/metabolismo , Animais , Transtornos da Coagulação Sanguínea/genética , Cicloeximida/farmacologia , Feminino , Meia-Vida , Técnicas In Vitro , Masculino , Microssomos Hepáticos/metabolismo , Biossíntese de Proteínas , Ratos , Fatores Sexuais , Deficiência de Vitamina K/sangue , Varfarina/farmacologiaAssuntos
Ligases/metabolismo , Microssomos Hepáticos/enzimologia , Oxirredutases/metabolismo , Vitamina K/metabolismo , Animais , Carbono-Carbono Ligases , Ácidos Cólicos , Ditiotreitol/farmacologia , Ativação Enzimática/efeitos dos fármacos , Compostos de Epóxi , Glutamatos , Cinética , Masculino , Polietilenoglicóis , Ratos , Solubilidade , Especificidade por SubstratoRESUMO
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.
Assuntos
Fígado/metabolismo , Vitamina K , Varfarina , Animais , Resistência a Medicamentos , Éteres Cíclicos/metabolismo , Feminino , Masculino , Necessidades Nutricionais , Oxirredutases/metabolismo , Ratos , Fatores Sexuais , Especificidade da Espécie , Vitamina K/análogos & derivados , Vitamina K/metabolismoRESUMO
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.
Assuntos
Ácidos e Sais Biliares/metabolismo , Colesterol/metabolismo , Animais , Ácidos e Sais Biliares/urina , Ductos Biliares , Radioisótopos de Carbono , Cateterismo , Ácido Quenodesoxicólico/metabolismo , Ácidos Cólicos/análogos & derivados , Ácidos Cólicos/metabolismo , Cromatografia Gasosa , Cromatografia em Camada Fina , Ácido Desoxicólico/metabolismo , Ácido Litocólico/metabolismo , Masculino , Ratos , Fatores de TempoAssuntos
Ácidos e Sais Biliares/análise , Cromatografia Gasosa , Fenômenos Químicos , Química , Ésteres , Éteres , Métodos , Silício , Análise Espectral , Fatores de TempoRESUMO
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.