No abstract

Binding proteins for weak organic acids, in particular bile acids and loop diuretics, were separated by two-dimensional gel electrophoresis after photoaffinity labelling of rat liver basolateral plasma membranes. After staining with Coomassie Blue the protein spots were isolated, electroeluted and concentrated in an SDS gel. The proteins were digested in the gel by endoproteinase Lys-C. Appropriate fragments were separated by HPLC and the sequence was determined. A 57-kD protein with an apparent pi of 5.3, which was marked by photoaffinity labelling with 7,7-azotaurocholate and stained with an antibody against recombinant protein disulfide isomerase (PDI), showed complete identity with PDI in three fragments. [³H]bumetanide, a loop diuretic, marked two 54-kD proteins. One 54-kD protein with an apparent pi of 6.35 showed homology to the tyrosine kinase inhibitor of the insulin receptor. The amino acid sequence of three fragments belonging to the other 54-kD protein with an apparent pi of 6.2 was identical with cytokeratin 8. Two further proteins that were present in large amounts were also sequenced. A 35-kD protein was identified to be arginase. The internal sequences of a 58-kD protein showed 100% homology to endoproteinase ER 60. The results show that the proposed method of isolation of proteins from 2D gels (a) provides sequence data, even if there are only minute amounts of protein obtainable, and (b) allowed identification of yet unknown binding proteins of organic anions in the liver.

A hepatic bumetanide‐binding protein of molecular mass 60 kDa was isolated from rat liver sinusoidal plasma membranes after photoaffinity labelling with [3H]bumetanide. The protein was purified by non‐equilibrium pH gel electrophoresis/two‐dimensional gel electrophoresis. The amino acid sequences of two internal fragments share 67% and 89% similarity with rat liver catalase, which has a molecular mass of 59.758 kDa. With H2O2 as a substrate, the catalytic activity was measured in rat liver plasma membrane preparations. This activity was blocked by bumetanide and aminobumetanide. Polyclonal antibodies were raised against the purified 60‐kDa membrane bumetanide‐binding protein. The antibody anti‐Bum‐Ab 60 immunoprecipitated a 60‐kDa protein from rat hepatocytes. Immunoblot analysis of SDS/PAGE and two‐dimensional PAGE gels confirmed that the antibody was specific for the 60‐kDa bumetanide‐binding protein and cross‐reacted with commercially available purified bovine liver catalase. Immunofluorescence showed the presence of the 60‐kDa antigen in the plasma membrane of intact hepatocytes. Western‐blot analysis revealed that the protein was present in rat kidney cortex homogenate but was lacking in hepatoma cells AS‐30 D, Reuber H35 FAO and HPCT cells (clone 1E3), in spleen, and in ileum. These results indicate that a plasma‐membrane‐derived catalase binds bumetanide in rat liver.

A hepatic bumetanide-binding protein of molecular mass 60 kDa was isolated from rat liver sinusoidal plasma membranes after photoaffinity labelling with [3H]bumetanide. The protein was purified by non-equilibrium pH gel electrophoresis/two-dimensional gel electrophoresis. The amino acid sequences of two internal fragments share 67% and 89% similarity with rat liver catalase, which has a molecular mass of 59.758 kDa. With H2O2 as a substrate, the catalytic activity was measured in rat liver plasma membrane preparations. This activity was blocked by bumetanide and aminobumetanide. Polyclonal antibodies were raised against the purified 60-kDa membrane bumetanide-binding protein. The antibody anti-Bum-Ab 60 immunoprecipitated a 60-kDa protein from rat hepatocytes. Immunoblot analysis of SDS/PAGE and two-dimensional PAGE gels confirmed that the antibody was specific for the 60-kDa bumetanide-binding protein and cross-reacted with commercially available purified bovine liver catalase. Immunofluorescence showed the presence of the 60-kDa antigen in the plasma membrane of intact hepatocytes. Western-blot analysis revealed that the protein was present in rat kidney cortex homogenate but was lacking in hepatoma cells AS-30 D, Reuber H35 FAO and HPCT cells (clone 1E3), in spleen, and in ileum. These results indicate that a plasma-membrane-derived catalase binds bumetanide in rat liver.