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Jul 29, 2020
THN JOURNAL OF BIOLOGICAL CAEMIBTRY Vol. 249, No. 11, Iseue of June 10, pp. 3823-3337, 1974
P&&d in U.S.A.
A POTENT INHIBITOR OF MITOCHONDRIAL OXIDATIVE PHOSPHORYLATION*
(Received for publication, November 12, 1973)
ADRIAN R. L. GEAR
From the Department of Biochemistry, The University of Virginia, Charlottesville, Virginia ,%Y?901
The lipophilic dye, rhodamine 6G, has been shown to be a potent inhibitor of oxidative phosphorylation, with a Ki of about 3 w, corresponding to 1.2 nmoles of dye per mg of mitochondrial protein. ATP-supported Cazf accumulation was blocked, but not -that driven by succinate oxidation. Concentrations of rhodamine 6G above 20 PM slowly un- couple respiration and inhibit respiration-dependent Ca2+ uptake. Neither the dinitrophenol-stimulated ATPase nor uncoupled respiration of intact mitochondria were inhibited by the dye, even at 50 PM. Arsenate-stimulated respiration, on the other hand, was partially blocked by 7.5 PM dye, but energy-linked phosphate accumulation was unaffected. Be- low 10 PM rhodamine 6G, no mitochondrial swelling, loss of matrix protein, or endogenous I(+, Cazf, or Mg2+, was ob- served. The dye was bound very tightly to mitochondria, being present at 1.3 and 0.8 nmoles per mg of protein for the inner and outer membranes, respectively. Binding was also monitored by direct measurement of H+ release into the suspending medium in the presence of rhodamine 6G. Some 0.6 to 0.75 mole of Hf was ejected per mole of dye added, of which only about 50% was firmly bound. A red shift occurred on binding, the X,,, increasing from 527 to 535 nm. The related compound, rhodamine B, a free acid and
uncharged at pH 7, was completely without influence on mitochondrial energy-linked functions.
Kinetic studies on the rate of ADP-stimulated Hf uptake in the presence and absence of rhodamine 6G yielded non- linear, Lineweaver-Burk plots and were of noncompetitive type. The K,,, for ADP decreased from 56 to 29 PM in the presence of 4 PM rhodamine 6G. When the data is expressed as a Hill plot, straight lines are obtained, with the value of e, the interaction coe5cient, increasing from 1.28 to 1.72 after dye addition.
Rhodamine 6G did not inhibit the Mg2+ATPase in soni- cated submitochondrial particles, unlike aurovertin. It did, however, block adenine nucleotide binding to intact mito- chondria, both for [ICJATP and [14C]ADP. Inhibition by 10 PM dye for low levels of added nucleotide was identical with that caused by 10 PM atractyloside, but was only very slight at higher nucleotide concentrations (0.2 mM).
The results of this study support the conclusion that rhodamine 6G blocks the adenine nucleotide translocase apparently being similar to atractyloside and bongkrekic acid. However, in sharp contrast to these inhibitors, rhodamine 6G did not inhibit the 2,4-dinitrophenol- stimulated ATPase of intact mitochondria. Consequently, uncouplers destroy the dye’s ability to interfere with the translocase, and this suggests a lipid binding to be involved in the action of rhodamine 6G.
This paper describes the action of the lipophilic dye, rhodamine 6G on mitochondrial energy-linked functions. The compound is positively charged at pH 7, compared to its close relative,
* This research was supported by the United States Public Health Service National Institutes of Health Grant GM-01814, as well as a subgrant from Institutional Grant (Gu-2551) to the University of Virginia by the National Science Foundation.
Rhodamine B rhodamine B, which is not, due to the presence of a free carboxyl group. Some studies on rhodamine 6G by Huang el al. (1) pointed to specific binding with well characterized liposome preparations. In addition, Levshin and Baranova (2) have described the detailed titration behavior of rhodamine 6G. These observations helped prompt the present investigation of lipophilic dyes like rhodamine 6G on energy-linked functions of
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mitochondria, especially since phospholipids probably play a key role in energy transduction (3).
It was found that the dye strongly inhibits both oxidative phosphorylation and ATP-supported ion transport, while having little action on respiration driven cation uptake by mitochondria. The Ki for inhibition of oxidative phosphorylation was about 3 PM. Interestingly, 2,4-dinitrophenol-stimulated ATPase was not inhibited at all. Concentrations above 10 PM began to un- couple respiration and inhibit respiration-driven cation transport. Based upou these studies as well as ou ATPase activity and arsenate-stimulated respiration, it was concluded that rhodamine 6G probably acts on the adenine nucleotide translocase. Direct measurement of this revealed very strong inhibition of adenine nucleotide binding at low, but not at high nucleotide levels. Lack of inhibition of the Mg2fATPase in “inside-out” submito- chondrial particles also points to nucleotide translocase involve- ment. It was also evident that a uet positive charge is essential for inhibition, since rhodamine B, which is uncharged at pH 7, was completely without effect on oxidative phosphorylation.
Rhodamine GG was kindly supplied and purified chromato- graphically by Dr. C. Huang of the I)epartment of Biochemistry, University of Virginia. Rhodamine B was obtained from the Aldrich Chemical Co. and also made up to 0.2 rnM in deionized water. Oligomycin and atractyloside were supplied by the Sigma Chemical Co., St. Louis, MO., and dissolved in ethanol at 2 mg per ml. Lubrol WX, a nonionic detergent, came from I.C.I. Organic.3 Inc., Provincetown, R.I. Gramicidin, digitonin, and m-chlorocarbonylcyanide phenylhydrazone were from Mann Re- search Chemicals. 46CaC12, [idC]ADP, and [idC]ATP were ob- tained from Amersham-Searle, Chicago, 111. All other reagents were of AR grade, or the highest purity commercially available.
Apparatus An oxygen electrode (Yellow Springs Instrument Co.) in a
water-jacketed, closed chamber (Gilson Electronics, Madison, Wis.), was used to monitor mitochondrial respiration and oxida- tive phosphorylation. Hydrogen ion movements, both for ion uptake and oxidative phosphorylation and ATPase studies, were followed with a microcombination electrode (Thomas, 4858-L15) coupled to a Beckman Expandomatic pH meter and a Honeywell 194, or Sargent SLR 10” recorder. Potassium movements were monitored with a Beckman cation No. 39047 electrode used in conjunction with an Orion No. 90-02 reference electrode, and dis- played with the above recording system.
Male albino Sprague-Dawley rats were obtained from Carworth Inc., New York, N.Y. or from Charles River Breeding Labora- tories, Wilmingt,on, Mass.; Wistar strain rats were obtained from Mann Research in Puerto Rico (now Purina-Ralston Laboratory Animals, Vincent Town, N.J.). The animals weighed from 150 t,o 300 g and were kept on normal rat chow until killed.
TABLE I Comparison of phosphate release and H+ ejection during
assay of 2,4-dinitrophenol-stimulated ATPase Rat liver mitochondria, 2.5 mg per ml, were incubated in a
medium containing 80 mM NaCl, 10 mM MgC12, 10 mM sodium succinate, and 5 mM Tris-Cl, pH 7.4. One minute after the addi- tion of ATP at 0.25 mM, 5 X 10-c M 2,4-dinitrophenol was used to initiate ATPase action. Recording of pH was continuous and every 10 s up to 1 min, l-ml aliquots were withdrawn for phos- phate analysis. After that, phosphate analyses were done every minute up to 5 min. Results are expressed as the mean ratio of H+ to PO4 over the two time periods and include standard devia- tions.
Initial 1 min Final 4 min
H+/PO, 1.003 =t 0.148 1.033 f 0.072
branes, the mitochondria were subjected to digitonin treatment (6). High amplitude, respiration-dependent swelling was moni- tored spectrophotometrically by following the extinction changes at 520 nm with a Gilford recording spectrophotometer (7). Res- piration and ATP-supported calcium uptake were determined by pH and isotope procedures previously described (8). Oxidative phosphorylation was studied both by pH and polarographic tech- niques (5).
ATPase Assay by Monitoring pH-ATPase activity whether 2,4-dinitrophenol or Mg2+-stimulated, was monitored by following pH changes instead of by the more common assay of phosphate or ADP. The pH technique is particularly suited for measuring rapid, initial rates of ATP hydrolysis, but becomes less desirable for reactions longer than 10 min; mainly, because of the slow pH drift, which has to be allowed for. This problem can be minimized by increasing the buffering power of the medium.
In order to validate the use of H+ for assaying ATPase activity, the following experiments were performed. First, 2,4-dinitro- phenol-stimulated ATPase was followed over 5 min. During the 1st min, five samples were withdrawn for phosphate analysis, and then one sample every minute for 4 min. The results of experi- ments on three different mitochondrial preparations are given in Table I. Very good agreement between the two parameters, H+ and Pod, is evident.
A second experiment was carried out, using a pa-stat titrator (Radiometer, Copenhagen) to show that the small pH changes during a pH experiment do not lead to false H+ movements. The latter might conceivably arise due to pK effects and change in buffering power with pH. In any event the total change in pH during an actual ATPase ass