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Use of ³¹P NMR to Assess Effects of DNP on ATP Levels in Vivo in Barley Roots

United States Department of Agriculture, Agricultural Research Service, Plant Physiology Institute, Beltsville Agricultural Research Center, Beltsville, Maryland 20705, Physical Chemistry and Instrumentation Laboratory, Eastern Regional Research Center, 600 East Mermaid Lane, Philadelphia, Pennsylvania 19118

Previous work has shown that undissociated 2,4-dinitrophenol (DNP) both increases the permeability of roots to ions and alters the membrane lipids of barley roots. Anionic DNP is the main entrant form but has no effect on permeability or on the membrane lipids. The amount of anionic DNP taken up by the roots is sufficient, that were it in free solution in the cytoplasm, the DNP would uncouple oxidative phosphorylation, and thereby inhibit ATP synthesis. The present work was undertaken to assess whether DNP alters ATP levels when it is taken up by barley roots. ³¹P nuclear magnetic resonance spectra were used to monitor, in vivo, levels of ATP, cytoplasmic phosphate, vacuolar phosphate, and other phosphate compounds in barley roots in the presence of 10 micromolar DNP at pH 5 and pH 7. The spectra indicate that no change in the level of ATP or the cytoplasmic pH occurred in the roots in the presence of DNP for as long as 20 hours. Thus, the effects of undissociated DNP are effects directly on the root membranes and do not involve inhibition of ATP synthesis. Furthermore, the results explain why anionic DNP has no effect on ion uptake and accumulation.