A spontaneous mutant of Methanothermobacter thermautotrophicus resistant toward the ATP-synthase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD) was isolated. DCCD normally inhibits methanogenic electron-transport-driven ATP synthesis, however, the DCCD-resistant strain exhibited methanogenesis in the presence of 300 micromol/L DCCD. Total ATP synthesis was shown to be higher in the mutant strain, both in the presence and absence of DCCD. These results suggested a modification in the ATP-synthesizing system of the mutant strain. Using Blue Native PAGE combined with MALDI TOF/TOF mass spectrometry, increased concentrations of both the A(1) and A(o) subcomplexes of the A(1)A(o)-type synthase were identified in the mutant strain. However, no alterations were found in the structural genes (atp) for the A(1)A(o) ATP synthase. The results imply that DCCD resistance is a consequence of increased A(1)A(o) ATP synthase expression, and suggest that genes involved in regulating synthase expression are responsible for DCCD resistance.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• archeální proteiny biosyntéza   MeSH
• dicyklohexylkarbodiimid toxicita   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• exprese genu MeSH
• inhibitory enzymů toxicita   MeSH
• léková rezistence * MeSH
• methan metabolismus   MeSH
• Methanobacteriaceae chemie   účinky léků   izolace a purifikace   metabolismus   MeSH
• mutace * MeSH
• oxidace-redukce MeSH
• protonové ATPasy biosyntéza   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• upregulace MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• archeální proteiny MeSH
• dicyklohexylkarbodiimid MeSH
• inhibitory enzymů MeSH
• methan MeSH
• protonové ATPasy MeSH

The biochemical basis of a defective bioenergetic system was attempted to be determined in N,N'-dicyclohexylcarbodiimide (DCCD)-resistant mutant of Methanothermobacter thermautotrophicus. Components participating in the maintenance of methanoarchaeal membrane structure and function, such as the composition of the mixture of squalene and its hydrosqualene derivatives and also properties of membrane-associated proteins were compared in wild-type and mutant cells. The impairment of the bioenergetic system in DCCD-resistant mutant was detectable in the membrane-protein profile; it was also accompanied by changes in proportions of squalene-hydrosqualenes.

• MeSH
• antibiotická rezistence * MeSH
• buněčná membrána metabolismus   MeSH
• dicyklohexylkarbodiimid metabolismus   farmakologie   MeSH
• energetický metabolismus MeSH
• membránové proteiny genetika   metabolismus   MeSH
• Methanobacteriaceae účinky léků   genetika   metabolismus   MeSH
• mutace MeSH
• skvalen chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dicyklohexylkarbodiimid MeSH
• membránové proteiny MeSH
• skvalen MeSH

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.

• MeSH
• amilorid farmakologie   MeSH
• bakteriální léková rezistence účinky léků   genetika   fyziologie   MeSH
• bakteriální proteiny chemie   účinky léků   MeSH
• blokátory sodíkových kanálů farmakologie   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• membránové proteiny chemie   účinky léků   genetika   MeSH
• Methanobacteriaceae účinky léků   genetika   fyziologie   MeSH
• mutantní proteiny chemie   účinky léků   MeSH
• Na(+)-H(+) antiport chemie   účinky léků   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• amilorid MeSH
• bakteriální proteiny MeSH
• blokátory sodíkových kanálů MeSH
• membránové proteiny MeSH
• mutantní proteiny MeSH
• Na(+)-H(+) antiport MeSH