THE 60-KDA BUMETANIDE-BINDING PROTEIN FROM RAT-LIVER MEMBRANES IS A CATALASE

A hepatic bumetanide-binding protein of molecular mass 60 kDa was isol ated from rat liver sinusoidal plasma membranes after photoaffinity la belling with [H-3]bumetanide. The protein was purified by nonequilibri um pH gel electrophoresis/two-dimensional gel electrophoresis. The ami no acid sequences of two internal fragments share 67% and 89% similari ty 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 L iver plasma membrane preparations. This activity was blocked by bumeta nide and aminobumetanide. Polyclonal antibodies were raised against th e purified 60-kDa membrane bumetanide-binding protein. The antibody an ti-Bum-Ab 60 immunoprecipitated a 60-kDa protein from rat hepatocytes. Immunoblot analysis of SDS/PAGE and two-dimensional PAGE gels confirm ed that the antibody was specific for the 60-kDa bumetanide-binding pr otein and cross-reacted with commercially available purified bovine li ver catalase. Immunofluorescence showed the presence of the 60-kDa ant igen in the plasma membrane of intact hepatocytes. Western-blot analys is revealed that the protein was present in rat kidney cortex homogena te 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 tha t a plasma-membrane-derived catalase binds bumetanide in rat liver.