Amiloride resistance in the methanoarcheon Methanothermobacter thermoautotrophicus: characterization of membrane-associated proteins.
An amiloride-resistant mutant with diminished Na+/H+ antiporter activity was isolated from Methanothermobacter thermoautotrophicus. To define the protein basis of amiloride resistance, the composition of membrane-associated proteins was partially characterized and compared with that of the wild type strain. An abundant 670-kDa membrane-associated protein that was present only in the mutant strain was analyzed by MALDI-TOF MS and identified as a coenzyme F420-reducing hydrogenase. The amiloride resistance was not accompanied by changes in protein size or changes in the level of subunits A or B of the A1A0-type ATP synthase; on the other hand, the SDS-PAGE patterns of the chloroform-methanol extract of membranes from both strains were different. Two bands with calculated molecular mass 16 and 11 kDa were identified as MtrD and AtpK, respectively. The observed over-expression of a 22.7-kDa protein in the mutant cells may represent the multimeric form of the MtrD subunit. These results show that the impairment of the Na+/H+ antiporter system in the amiloride-resistant mutant of Methanothermobacter thermoautotrophicus is accompanied by only small changes in a few membrane-associated proteins.
Surín
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Institute of Animal Biochemistry and Genetics, Slovak Academy of Sciences, Ivanka pri Dunaji, Slovakia.
Cubonová
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Majerník
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AI
Smigán
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D000584:Amiloride / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000187:drug effects; D024881:Drug Resistance, Bacterial / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D008565:Membrane Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D017013:Methanobacteriaceae / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D050505:Mutant Proteins / Q000737:chemistry / Q000187:drug effects; D026941:Sodium Channel Blockers / Q000494:pharmacology*; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000187:drug effects; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D001426:Bacterial Proteins; D008565:Membrane Proteins; D050505:Mutant Proteins; D026941:Sodium Channel Blockers; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride