RESUMO
Protein disulfide isomerase (PDI) was considered to be involved in the hepatic uptake of certain organic anions because the protein is photoaffinity labeled by photolabile derivatives of the bile acid taurocholate. Several lines of evidences including photoaffinity labeling experiments indicated a close relationship between the uptake of bile acids and the organic anion bumetanide. The possible involvement of PDI in hepatic transport processes of these organic anions was tested with polyclonal antibodies raised against a PDI-beta-galactosidase fusion protein. Western blot analysis and immunofluorescence of intact hepatocytes showed that protein disulfide isomerase is located in sinusoidal rat liver plasma membranes. This protein is immunologically identical with microsomal PDI prepared from bovine liver. The plasma membrane form of PDI is, however, not labeled by photoactivated bumetanide as revealed by two-dimensional gel electrophoresis. These results indicate that, although a membrane-bound form of the PDI is present in the sinusoidal plasma membrane of rat hepatocytes, this protein is not involved in the hepatocellular uptake of the organic anion bumetanide.
Assuntos
Isomerases/metabolismo , Fígado/enzimologia , Animais , Ânions/metabolismo , Anticorpos , Western Blotting , Bovinos , Membrana Celular/enzimologia , Cromatografia por Troca Iônica , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Isomerases/análise , Isomerases/isolamento & purificação , Cinética , Peso Molecular , Isomerases de Dissulfetos de Proteínas , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , beta-Galactosidase/biossíntese , beta-Galactosidase/isolamento & purificaçãoRESUMO
By affinity labeling with photolabile [3H]bumetanide, a 52-54 kDa bumetanide binding protein was identified in the sinusoidal plasma membrane fraction from rat liver. The protein is assumed to represent the carrier for hepatic uptake of loop diuretics. By two-dimensional (2D) gel electrophoresis we have purified this protein from hepatocytes, sinusoidal plasma membranes and subfractions of associated and integral plasma membrane proteins. Amongst more than 20 protein spots, a single integral plasma membrane protein was detected. The apparent pI of this molecule is 6.7. Specific labeling of this protein was not found in the fraction of associated plasma membrane proteins. To detect possible binding of radioactive bumetanide to microsomal cytochrome P450s, photolabeling experiments with integral plasma membrane proteins were performed under nitrogen/carbon monoxide atmosphere and in the presence of piperonyl butoxide. Labeling of the 52-54 kDa protein was not affected by these inhibitors of P450 enzymes. Taken together, these results indicate that the bumetanide binding protein is very likely to be a non-microsomal integral plasma membrane protein.