The 60-kDa bumetanide-binding protein from rat liver membranes is a catalase.
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.
Ottallah-Kolac
M
M
Institute of Pharmacology and Toxicology, Justus-Liebig-Universität, Giessen, Germany.
Tripier
D
D
Brühl
B
B
Platte
H D
HD
Jouvenal
K
K
Schuh
K
K
Kemmer
H
H
Petzinger
E
E
D000595:Amino Acid Sequence; D000818:Animals; D002034:Bumetanide / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002374:Catalase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry; D003429:Cross Reactions; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D007672:Kidney Cortex / Q000737:chemistry; D008099:Liver / Q000737:chemistry*; D008297:Male; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D051381:Rats; D017208:Rats, Wistar; D028021:Sodium-Potassium-Chloride Symporters
D002352:Carrier Proteins; D008565:Membrane Proteins; D028021:Sodium-Potassium-Chloride Symporters; D002034:Bumetanide; D002374:Catalase