Functioning of the Adenine Nucleotide Transporter in the Arsenate Uncoupling of Corn Mitochondria

ADP (9,13, 15,26). As in animal mitochondria (6), Val plus K+ will activate plant mitochondrial ATPase, being fully as effective as 2,4-dinitrophenol (12). To an impressive degree, Val plus K+ produces uncoupling of plant mitochondria resembling that obtained with the classical, proton-conducting uncouplers.Rat liver (18) and beef heart (4) mitochondria are believed to possess antiporters for exchanging Na+ for H+, with much less activity in K+/H+ exchange. It has been suggested (P. Mitchell, private communication) that the release of respiration in corn mitochondria with little swelling (9) could arise from the activity of a K+/H+ antiporter, and that such an antiporter may be characteristic of plants. It is characteristic of plants that Na+ is not an essential element (some halophytes excepted), and it is reasonable to speculate that an active K+/H+ antiporter exists.We report here an investigation of Val-uncoupled respiration of corn mitochondria as related to influx and efflux pumping of phosphate salts. (The term "pumping" is used to denote energy-linked salt transport in or out of the matrix.) It seems that a K+/H+ antiporter may in fact exist, but that it is not really K+ specific. Specificity seems to lie primarily with a lipid barrier which has high resistance to cation penetration unless ionophores are used. Uncouplers in turn may promote penetration of protons to this cation+/H+ antiporter.Valinomycin is widely used as a lipid-soluble, potassiumbinding ionophore for rapidly equilibrating the electrochemical potential of K+ across membranes. With animal mitochondria this equilibration does not effectively uncouple respiration unless an anion (e.g., phosphate or acetate) is present which can be transported at the expense of a proton gradient, in which case there is active swelling (19,20). If permeability to chloride is induced, respiration is released by ValP in active KCI extrusion (3). With low K+ concentrations, Val does not eliminate respiratory control or phosphorylation of liver mitochondria (11,17,21).Plant mitochondria are at least quantitatively different. There is rapid passive swelling in KCI at neutral pH which is accelerated by gramicidin D (15, 27) or Val (13,25), demonstrating permeability to chloride which is rate limited by accompanying cation influx. Efflux pumping of the KCI with concomitant shrinkage is not uncoupled by Val although respiration is accelerated (13). With sucrose or mannitol- MATERIALS AND METHODSCorn mitochondria (Zea mays L., WF9-Tms X M14) were isolated as previously described (8) in 0.4 M sucrose, 50 mM KH,PO, 5 mm EGTA, adjusted to pH 7.6 with tris. In certain cases endogenous K+ was reduced by substituting NaH,PO4 as buffer; this lowered the endogenous K+ from 172 to 78 nmoles/mg of protein. Potassium was determined by flame emission spectroscopy on 5% trichloroacetic acid extracts.Reactions were run in 4-ml volumes at 29 C with simultaneous recordings of 02 and per cent transmission as previously described (8, 9), except that full scale transmittance ...

Section: Methodssupporting

confidence: 50%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

The Action of Valinomycin in Uncoupling Corn Mitochondria

Hensley¹,

Hanson²

1975

“…This is accomplished by having the ATPase (indicated as E) bind ATP furnished from the cytoplasm. Bound adenine nucleotides are known for animal mitochondrial ATPase (8) and they may function in ATP formation (3); our laboratory has suggested a role for them in corn mitochondrial ATPase (2). Hydrolysis in the plasmalemma is from the solution side, producing bound ADP and Pi, and releasing H+ to the solution (Fig.…”

Section: Discussionmentioning

confidence: 99%

Cell Potentials, Cell Resistance, and Proton Fluxes in Corn Root Tissue

Lin¹,

Hanson²

1976

“…the large increase in 32Pi uptake induced by rotenone with only a small increase in [3H]ADP uptake (Table I) Table V. (Table II), which is to be expected on the basis of its ready transport via the phosphate transporter (5). Sulfate shows a little activity, and again this might be expected since sulfate in the absence of phosphate enters on the phosphate transporter, but with low efficiency (4).…”

Section: Discussionmentioning

confidence: 96%

Energy-linked Adenosine Diphosphate Accumulation by Corn Mitochondria

Abou‐Khalil¹,

Hanson²

1979

In the preceding papers (1-3) we have described the general properties of ADP accumulation by corn mitochondria. The transport mechanism is insensitive to C-ATR,2 the inhibitor of the AdN translocase, but is very sensitive to inhibitors of phosphate transport. Phosphate and Mg2+ are required for ADP accumulation, and questions arise as to the nature of this requirement. We report here on the specificity of the transport system for these ions, on their accumulation during ADP uptake, and on the possibility that ADP enters in exchange for phosphate. We speculate that the Mechanism of ADP accumulation in corn mitochondria is one of concerted phosphate + divalent cation + ADP uptake. The Millipore filtration procedure for determining [3HJADP uptake was as described (1). The same procedure was used for 32Pi uptake. Millipore filters were presoaked in unlabeled 2 mm ADP and/or 5 mm Pi to reduce the radioactivity absorbed by the filter from the medium. MATERIALS AND METHODSFor determining if accumulated phosphate would exchange for ADP, the mitochondria were preloaded with 'Pi by incubation at room temperature for 11 min with about 12 mg protein in 9 ml of the basic medium containing 10.7 mM K 32Pi, 15 ,Ig oligomycin, 60 Amol NADH, and 4 mm MgCl2. The mitochondria were collected by rapid centrifugation of 0.8-ml aliquots in an Eppendorf 5412 centrifuge; the pellets were rinsed with cold 0.2 M sucrose, and then resuspended in 0.8 ml basic medium in the centrifuge tubes containing oligomycin, NADH, and MgCl2 at the same concentrations used during loading. Additions were made as indicated in Figure 2, and after 3-min incubation the mitochondria were collected by rapid centrifugation. The final pellets were dissolved in formic acid and counted as previously reported (18).The Mg2" content of mitochondria was determined by using a Jarrell-Ash model 82-700 atomic absorption spectrometer. Extracts of mitochondria were collected in 12% trichloroacetic acid by using the silicone oil method as described (3 [3H]ADP and 32Pi were purchased from New England Nuclear, C-ATR from Boehringer, nucleotides, Tes, and BSA from Sigma, and all other reagents were analytical grade.