MEMBRANE POTENTIALS IN PLANT MITOCHONDRIA

The positively charged dye, safranine, has been used as an indicator of membrane potentials in mung bean (Phaswois aureus) and Voodoo lily (Sauromatwm gutatum) mitochondria under a variety of metabolic conditions. The spectral response of safranine has been calibrated with respect to a K' diffusion potential and was found to be linearly related to the developed potential within the range of50 to 160 millivolts. Both respiration and ATP hydrolysis gave rise to a membrane potential of approximately 135 millivolts. Respiratory inhibitors such as cyanide and antimycin depolarized the potentiaL whereas rotenone has little effect. No potentials were developed during NADH supported cyanide insensitive respiration. It is concluded that safranine may be a useful spectrophotometric probe of the mitochondrial membrane potential. It is generally accepted that in the energized state, mitochondria generate a large membrane potential (A+) (6, 8). Measurements of such membrane potentials are based mainly on the distribution of permeant ions and are thought to provide reliable indications of AM under steady-state conditions (19). Recently, however, attention has been focused on the use of extrinsic probes as indicators of membrane potential (4) in both cells (2) and organelles (1, 3). The use of such probes has several advantages over ion distribution methods. In particular, experimental procedures are simple, information is obtained rapidly and continuously, and, because such compounds may be used in micromolar concentrations, they cause minimal interference to the system under investigation. In the present study, we have used the positively charged dye, Safranine 0 as an indicator of membrane potential in intact plant mitochondria and investigated its response under a variety of metabolic conditions. It was found that the extent ofthe spectral shift in safranine absorbance was linearly related to the developed membrane potential and could be calibrated with reference to a K+-diffusion potential. The linearity of the shift was, however, affected by variations in dye:protein ratios. The present study indicates a qualitative correlation between AI and safranine response and suggests that the probe may be a useful indicator of changes in membrane potential in intact plant mitochondria. ' Supported by grants from the National Science Foundation (W. D. B.) and S. E. R. C. (A. L. M.). 2 To whom correspondence should be addressed. 'Abbreviations: FCCP, p-trifluoromethoxy carbonylcyanide phenylhydrazone; SHAM, salicylhydroxamic acid. MATERIALS AND METHODS Preparation of Mitochondria. Mitochondria were isolated from etiolated mung bean hypocotyls (Phaseolus aureus) or the spadices of Voodoo lily (Sauromatum guttatum) by the general method of Bonner (5), with adaptions as previously described (9). Oxygen Consumption. Mitochondrial respiration was measured polarographically in 2 ml of reaction medium containing 0.3 M mannitol, 5 mM MgCl2, 10 mm KCI, and 10 mm K-phosphate (pH 7.2) (unless otherwise indicated) using a Rank O2 electrode (Rank Bros., Cambridge, U.K.) at 250C. Spectrophotometric Techniques. A Johnson Research Foundation dual wavelength spectrophotometer was used to monitor changes in the absorption of safranine 0 using the wavelength pair 511 to 533 nm. Safranine 0 was purchased from Sigma, and used without further purification by addition of small aliquots of 2 mm aqueous solution of the dye to give a final concentration of 16 ,LM or approximately 25 nmol/mg protein. All experiments were performed with glass cuvettes of 1 cm light path, at room temperature, in 2.5 ml medium containing 0.3 M mannitol, 5 mm MgCl2,and 20 mm Hepes (pH 7.2). Further additions were made as indicated in the figure legends. Assays and Reagents. Protein was determined by the method of Lowry et al. (11) using crystalline BSA as a standard. Valinomycin, oligomycin, rotenone, and FCCP3 were purchased from Sigma and dissolved in absolute ethanol. Nigericin was a gift from Dr. J. C. Smith (Johnson Research Foundation). All other reagents were of the highest purity commercially available.