The 60-kDa bumetanide-binding protein from rat liver membranes is a catalase

• Published in: European journal of biochemistry Vol. 228; no. 2; pp. 506 - 514
• Main Authors: Ottallah-Kolac, M, Tripier, D, Brühl, B, Platte, H D, Jouvenal, K, Schuh, K, Kemmer, H, Petzinger, E
• Format: Journal Article
• Language: English
• Published: England 01.03.1995
• Subjects: Amino Acid Sequence; Animals; Bumetanide - metabolism; Carrier Proteins - isolation & purification; Carrier Proteins - metabolism; Catalase - antagonists & inhibitors; Catalase - isolation & purification; Catalase - metabolism; Cell Membrane - chemistry; Cross Reactions; Fluorescent Antibody Technique; Immunoblotting; Kidney Cortex - chemistry; Liver - chemistry; Male; Membrane Proteins - isolation & purification; Membrane Proteins - metabolism; Molecular Sequence Data; Molecular Weight; Rats; Rats, Wistar; Sodium-Potassium-Chloride Symporters
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1995.00506.x

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.