Uncoupling of Energy-Linked Functions of Corn Mitochondria by Linoleic Acid and Monomethyldecenylsuccinic Acid

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.The protonated (undissociated) form of DNP' increases the permeability of barley roots to ions (4) and simultaneously alters the membrane lipid composition (6). Anionic DNP is the main entrant form but has no effect on either ion uptake and permeability or the membrane lipids.

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Jackson¹,

Pfeffer²,

Gerasimowicz³

1986

“…In barley and corn mitochondria, competitive kinetics are shown by Pi and DNP (2,12), suggesting a direct abtack on I-X. Recently, van Regardless of mechanism, the net result will be as ill,ustrated in figure 3; hydrolysiis of I-X, recycling I and X for acceleraited electron flow.…”

Section: Discussionmentioning

confidence: 99%

“…Our observations on 'corn mitochondria (2,18,19,21) show 'that only high concentrations of uncoupler (e.g. 100-500 FM DN'P2) wi,ll accelerate spontaneous swelling and reduce contraction.…”

mentioning

confidence: 97%

Uncoupling of Respiration-Linked Contraction in Corn Mitochondria

Hanson¹,

Miller²,

Dumford³

1968

Self Cite

Abstract. Respiration-linked contraction of corn mitochondria is not noticeably reduced by low, uncoupling concentr*tions of dinitrophenol. However, if a oontraction/respiration ratio is calculated, the contraction proves to be uncoupled. Previous statements that contraction cannot be uncoupled from respiration are in error.The uncoupling of c-ontraction is oonsistent with the concept that dinitrophenol attacks a primary non.phosphoryla-ted high energy intermediate (I,.X). It is proposed thot this intermediate is linked to some oontracti-le mechanism such that the degree of contraction reflects the level of intermediate.One of the more perplexing aspects of respiration-linked contraction in swollen mitochondria is the ineffeotiveness of uncoupling agents on the process. Our observations on 'corn mitochondria (2,18,19,21) show 'that only high concentrations of uncoupler (e.g. 100-500 FM DN'P2) wi,ll accelerate spontaneous swelling and reduce contraction. In a phosphorylating medium, isutch concenftrations alimost eliminate oxidative phosphorylation and strongly inhibit the respiration. Low concentrations of uncoupler (e.g. 10-30 KM DNP) which are adequate 'to depress P/O ratios, accelerate ATPase, release acceptorless respiration, and reduce Ca24 + Pi transport, have little or no effect on respirationlinked contraction. The same in(senisitivity is fouindl with animal mitochondria (1,4, 7).We proposed that contraction is closely linked to respiration and cannot be reduced by uncoupling agents un'less they depress respiration (21). When evidence was obtained supporting our contention that contraction is associated wiith energy conservation, presumably as I-X, the hypothetical nonphosphorylated high energy intermediate, a scheme was presented relating contraction to -the level of Ir--(13).Recenitly we have re-examined the action of DNP on contraction and find our previoous statements to be in error. Dinitrophenol does uncouple contraction if expressed on a respiration b)asis comparable to the P/O ratio. Furthermore, net contraction can be reduced by DN'P w'hille the respiration rate is stilil very high. The findings 'Supported by grants from the Atomic Energy -Commi,ssion

“…DNP anions, which are the major species taken up by the roots, have no effect on ion uptake and retention, although DNP uncouples oxidative phosphorylation of barley root mitochondria (2,11). The uncoupling action of DNP is competitive with Pi.…”

Section: Discussionmentioning

confidence: 99%

Differences between Effects of Undissociated and Anionic 2,4-Dinitrophenol on Permeability of Barley Roots

Jackson

1982

Effects of 2,4-dinitrophenol (DNP) and several other substituted phenols on permeability of barley roots (Hordeum vulgare var. Trebi) to ions were assayed as a function of pH and phenol concentration. Solutions containing 0.1 micromolar undissociated DNP increase the permeability of barley root cells to small ions such as K+, Na+, Ca2+, and Cl with no inhibition of respiration. Undissociated forms of the other phenols increase permeability also, but they are less effective than DNP. Only the undissociated DNP is effective. Anionic DNP does not increase permeability or inhibit ion uptake, although it is the major species accumulated by the roots, both at pH 5 and pH 7. At pH 7, in contrast to pH 5, 10 micromolar DNP has no effect on ion permeability of barley roots yet it uncouples oxidative phosphorylation of barley root mitochondria. This indicates that the all too common use of DNP as a test for active transport or involvement of ATP synthesis can be misleading.DNP' is used often as an inhibitor to assess the extent to which a physiological process under study is "active" or involves ATP synthesis. DNP is well-known as an uncoupler of oxidative phosphorylation. However, it also increases cell membrane permeability to ions (4,15,18). The present work was undertaken to determine whether an inhibitor such as DNP is multifunctional. Inconsistency in the literature with respect to the concentration of DNP that is effective suggested that the effects of DNP on permeability may be those ofthe weak acid form. This is suggested also by effects on permeability of short chain aliphatic acids of which only the undissociated form is effective (14). Consequently, effects of DNP on the permeability of barley roots to ions were studied as a function of pH, concentration, and time. Effects of several other phenols were studied also, to assess the specificity of DNP effects on permeability. Uptake of 14C-labeled DNP was measured for comparison with the effects. MATERIALS AND METHODSThe roots were from 6-d-old seedlings of barley (Hordeum vulgare var. Trebi) which had been dark-grown in aerated 0.2 mm CaSO4 with or without 0.1 mM KCI at 22 to 25°C at pH 5.6. Roots were excised and rinsed several times with demineralized H20 just before the experiment. Roots