Open AccessThe Nature of the Electron Spin Resonance Signal during Aerobic Uptake of Mn 2+ in Mitochondria from Rat Liver
Author(s) -
BRAGADIN Marco,
POZZAN Tullio,
AZZONE Giovanni Felice
Publication year - 1983
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1983.tb07579.x
Subject(s) - electron paramagnetic resonance , pi , chemistry , phosphate , oligomycin , divalent , inorganic phosphate , electron transport chain , nuclear magnetic resonance , resonance (particle physics) , crystallography , analytical chemistry (journal) , biochemistry , enzyme , physics , chromatography , atpase , organic chemistry , particle physics
Rat liver mitochondria take up aerobically large amounts of divalent cations in the absence of exogenous phosphate. The electron spin resonance (ESR) spectrum of matrix Mn 2+ reveals the presence of two components: one, a sextet signal, corresponding to hydrated Mn 2+ ; another, a spin exchange signal, attributed either to Mn 2+ binding to specific high‐energy membrane sites or to complexes of Mn 2+ with inorganic phosphate. Identification of the spin exchange signal with a Mn‐P i complex is favoured by the evidence that the spin exchange signal is observed at pH 7.5 but not at pH 6.5 in the absence of exogenous P i, but at both pH 7.5 and 6.5 in the presence of exogenous P i. On the other hand both in the absence or presence of exogenous P i inhibition by N ‐ethylmaleimide of P i transport, abolishes the spin exchange signal. This signal is again observed when P i is generated in the matrix, in the presence of N ‐ethylmaleimide, by ATP hydrolysis, and again abolished by oligomycin. Finally, addition of uncouplers results in a very slow disappearance of the signal. The amount of Mn 2+ participating in the spin exchange signal has been calculated to be in the range of 50–60 nmol × mg protein −1 . This amount is compatible with the amount of endogenous P i present or generated in average mitochondrial preparations. The ESR spectrum obtained by superimposing the spectra of Mn 3 (PO 4 ) 2 precipitate and hydrated Mn 2+ , in appropriate concentrations and ratios, resembles closely the ESR spectrum during aerobic Mn 2+ uptake in mitochondria. The band width of the spin exchange signal of Mn 3 (PO 4 ) 2 is not constant and varies between 40 and 22 mT depending on the state of aggregation of the complex. The kinetics of aggregation can be followed in solution as a function of the concentration of Mn 2+ , P i and of pH. Similar kinetics can also be followed during aerobic Mn 2+ uptake by controlling the rate of Mn 2+ influx. The present data support the previous proposal [Pozzan et al. (1976) Eur. J. Biochem. 71 , 93–99] that the spin exchange singal is essentially due to a Mn 3 (PO 4 ) 2 precipitate in the mitochondrial matrix.