Open AccessIn vivo 31 P‐and 13 C‐NMR studies of ATP synthesis and methane formation by Methanosarcina barkeri
Author(s) -
SANTOS Helena,
FARELEIRA Paula,
TOCI René,
LeGALL Jean,
PECK Harry D.,
XAVIER António V.
Publication year - 1989
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1989.tb14663.x
Subject(s) - methanosarcina barkeri , protonophore , intracellular , extracellular , methanol , chemistry , intracellular ph , atp synthase , metabolism , biochemistry , methanogenesis , nuclear chemistry , biophysics , membrane , biology , enzyme , methane , organic chemistry
Carbon and phosphorus metabolism of cell suspensions of Methanosarcina barkeri strain MS (DSM 800), grown on methanol, were probed in vivo by NMR. The experimental conditions, which involved thick cell suspensions, did not significantly affect the efficiency of the rate of methanol uptake by cells. Following exposure to methanol an acidification of both the intracellular and the extracellular spaces was observed and a gradient of 0.5 pH units across the cytoplasmic membrane was determined from the 31 P‐NMR data. High levels of intracellular ATP up to 4 mM were detected. The ADP concentration determined in a suspension of starved cells was only 2 mM, suggesting that a significant amount of ADP may be immobilized and is thus not detectable by NMR. In the presence of the protonophore, 3.3′,4′,5‐tetrachlorosalicylanilide, the proton gradient was dissipated and the synthesis of ATP stopped. The inhibitor of the ATP synthase, N,N ′‐dicyclohexylcarbodiimide, was rather inefficient in inhibiting ATP synthesis. High concentrations of N,N ′‐dicyclohexylcarbodiimide (corresponding to 300 nmol/mg protein –1 ) were required to decrease the ATP content by approximately 60%, and, under these conditions, formation of acetyl phosphate was detected. However, the methanol consumption rate was not affected.