Effects of Ethidium Bromide on the Respiratory Chain and Oligomycin-sensitive Adenosine Triphosphatase in Purified Mitochondria from the Cellular Slime Mold Dictyostelium discoideum*

SUMMARY Mitochondria were isolated from the cellular slime mold, Dictyostelium discoideum, and partially purified by sucrose density gradient fractionation. The most purified mitochondrial fraction from the gradient contained essentially no contaminating lysosomes and minimal amounts of contaminating peroxisomes as determined by the marker enzymes N-acetyl-glucosaminidase and catalase. A mitochondrial fraction with the same amount of lysosomal and peroxisomal contamination was also isolated from cells which had been treated with ethidium bromide for 5 days. The most purified mitochondrial fraction from control and ethidium bro-mide-treated cells had an identical buoyant density of 1.181 to 1.182 g per ml, suggesting that treatment with the drug does not result in any drastic structural changes in the mitochondrial membrane which would affect its density. In the purified mitochondria from ethidium bromide-treated


Mitochondria
were isolated from the cellular slime mold, Dictyostelium discoideum, and partially purified by sucrose density gradient fractionation.
The most purified mitochondrial fraction from the gradient contained essentially no contaminating lysosomes and minimal amounts of contaminating peroxisomes as determined by the marker enzymes N-acetylglucosaminidase and catalase. A mitochondrial fraction with the same amount of lysosomal and peroxisomal contamination was also isolated from cells which had been treated with ethidium bromide for 5 days. The most purified mitochondrial fraction from control and ethidium bromide-treated cells had an identical buoyant density of 1.181 to 1.182 g per ml, suggesting that treatment with the drug does not result in any drastic structural changes in the mitochondrial membrane which would affect its density.
In the purified mitochondria from ethidium bromide-treated cells, the content of cytochromes u-u3 was decreased over 80% and that of cytochrome b by 35%. The activities of cytochrome oxidase and oligomycin sensitive ATPase were reduced approximately 80%, whereas the activity of succinate-cytochrome c reductase was decreased 50%. By contrast, the specific activities of NADH and succinate dehydrogenases were identical in the purified mitochondria from control and ethidium bromide-treated cells.
Previously, we had reported that the specific activities of these two enzymes had nearly doubled in whole cells maintained in ethidium bromide for a time equivalent to six or seven generations after growth had stopped (STUCHxLL, R. N., WEINSTEIN, B. I., AND BEAT-TIE, D. S. (1973) Fed. Eur. Biochem. Sot. Lett. 37,[23][24][25][26].
These results suggest that continued formation of new mitochondrial membranes, with an identical complement of succinate and NADH dehydrogenases, must occur despite the cessation of cell growth which occurs as a result of the ethidium bromide induced loss of mitochondrial enzymes.  (11) and mammalian cells in tissue culture (6-8) had suggested that ethidium bromide causes a primary effect on the formation of cytochromes u-u3 and a more gradual, perhaps secondary effect on the formation of cytochrome b. Presumably, the ability of the cells to grow and divide was lost when energy production by the respiratory chain became limiting due to the loss of these essential cytochromes.
1%~ contrast, the specific activity of the two mitochondrial membrane-bound flavoproteins, succinate and KADH dehydrogenases, gradually began to increase when the cells in ethidium bromide had stopped dividing.
The specific activity of both these enzymes had reached a value nearly twice that of the control cells after 5 days.
In order to investigate more fully these changes in the mitochondrial respiratory chain induced by ethidium bromide, especially the increase in the flavoprotein dehydrogenases, it was necessary to develop a method to obtain purified mitochondria from the slime mold.
A comparison of the activity of several enzymes in mitochondria obtained from both control and ethidium bromide treated cultures has indicated that treatment with the drug results in an 80% decrease in both cytochrome oxidase and oligomycin-sensitive ATPase, and a 50% decrease in succinatc-cytochrome c reductase.
No change in the activity of either succinate or NAL)H dehydrogenases in purified mitochondria was observed; however, the amount of mitochondrial protein per cell increased significantly.
These results are discussed in terms of different models for the control of mitochondrial biogenesis.

METHODS
Cell Culture-Axenic cultures of Diclyostelium discoideum strain Ax-3 were grown as described previously (10). Cells that were to be treated with cthidium bromide were grown to a density of 0.5 to 1.0 X lo6 cells ner ml before the addition of the drum (10 YB per ml, final concent;ation).
Usually 3 liters of cells were grown at 22-23" in a 4-liter Erlenmeyer flask wrapped with aluminum foil on a New Brunswick gyratory shaker at 200 rpm. The cells used as controls were in log phase (2 to 3 X lo6 cells per ml), since previous studies had indicated that slight variations in mitochondrial enzymatic activities occurred as the cells approached stationary phase (1 to 1.5 X lo7 cells per ml).
Preparelion of Mitochondria-Cells were harvested by pouring the growth media into 250.ml centrifuge bottles, accelerating the centrifuge to 4000 X g, and then shutting off the centrifuge. This procedure provided a rapid means of collecting cells from large volumes of growth media.
The pelleted cells were washed once in cold distilled water and then in a medium containing 0.25 M mannitol, 0.01 M Tris buffer (pH 7.6), and 0.001 M EDTA (Buffer A). For best results, a thick slurry of cells was pipetted into a 20 ml Thomas-Ten Brocck glass-glass homogenizer and given 8 to 20 twisting strokes by hand. The homogenate was then diluted with 3 volumes of Buffer A and centrifuged at 3000 X g for less than 1 min to collect unbroken cells.
The pellet was rehomogenized and recentrifuged at 3000 X g. The rehomogenization and recentrifugation procedure was repeated two more times. All of the supernatants were pooled and centrifuged at 750 X g for 10 min. The pellet was discarded and the supernatant was again centrifuged at 750 X g for 10 min.
The resulting supernatant was centrifuged at 7700 X q for 15 min.
The pellet was resuspended in Buffer A, gentlv homogenized. and centrifuged at 7700 X o for 15 min. The pellet was then diluted with Buffer A to give a protein concentration of 10 to 15 mg per ml. Three milliliters of the 7706 X g pellet fraction were carefully layered onto a 35 to 60% (w/v) preformed linear sucrose gradient (0.01 M Tris, pH 7.6, 0.001 M EDTA) which had been prepared the previous evening.
The gradients were then centrifuged in a Beckman SW 27 rotor at 27.000 mm for 105 min at 5". The gradients were fractionated by piercing the bottom of the tubes and collecting 20 fractions of approximately 1.6 ml each.
Density Determinalions-The refractive indices (sodium D line) of the sucrose gradient fractions were determined with a Bausch and Lomb (Abbe-type) refractometer at 25". The refractive indices were converted to densities from standard tables. Ewzyme Assays-N-Acetylglucosaminidase was assayed according to the procedure of Loomis (12) using fractions which had been lysed with 0.04'$!& Triton X-100.
A l-ml assay mixture containing 0.01 M sodium acetate, pH 5.0, 0.01 M p-nitrophenyl-N-acetyl-p-D-glucosaminide was incubated with the different fractions at 35" for appropriate times. The reaction was stopped by adding 1.0 ml of 1 M Na#ZOz and the absorbance at 420 nm was determined with a Gilford spectrophotometer.
One unit of activity is defined as the production of 1 nmol of p-nitrophenol per min under the above conditions. An absorbance coefficient of p-nitrophenol at 420 nm was measured to be 15.5 rnM-i cm-i. Cytochrome oxidase, succinate-cytochrome c reductase, succinate dehydrogenase, and NADH dehydrogenase were assayed at 25" in a Gilford spectrophotometer as described by Kim and Beattie (13).
Catalase was assayed according to the method of Luck (14). The change in absorbance at 240 nm was determined at 25". The absorbance coefficient of 43.5 rnM-r cm-l was used. ATPase activity was measured at 22" in a medium containing 3 mM MgCl?, 10 mM ATP, 50 mM Tris, pH 9.0, and approximately 1 mg of mitochondrial protein in a final volume of 1.0 ml as described by Kim and Beattic (13 and p-nitrophenyl-N-acetyl-p-n-glucosaminide grade III, were obtained from Sigma; proteose peptone and yeast extract were obtained from Difco; and sucrose (density gradient grade) from Schwarz-Mann.

Preparation of Purified &'itochondrial
Fraction-In our initial attempts to isolate mitochondria from the slime mold by differential centrifugation, the pellet obtained at any centrifugal force from 6000 to 9000 x g was heavily contaminated with lysosomes as determined by N-acetylglucosaminidase or acid phosphatase activity.
When the once washed 7700 x g pellet was further subjected to isopyknic centrifugation in a preformed continuous sucrose gradient, a singie band of protein was observed coincident with succinate dehydrogenase activity (Fig. 1) The refractive index for each fraction was measured as described under "Methods." mitochondrial fraction obtained by either differential or gradient centrifugation was calculated using marker enzymes for lysosomes and peroxisomes (Table I).
The mitochondria in both the 7700 x g pellet and in the gradient fraction of density 1.182 g per ml were significantly purified as indicated by the 9-and 17-fold increases in specific activity of succinate dehydrogenase compared to the homogenate, which in these experiments, is the 600 x g supernatant.
In addition, over 18% of the succinate dehydrogenase activity of the homogenate was recovered in the gradient fraction.
Although the specific activity of catalase was also increased 4-fold in this same gradient fraction, less than 5% of the homogenate activity was recovered in the most purified gradient fraction. Lysosomes, as indicated by the N-acetylglucosaminidase activity, were concentrated in the 7700 x g pellet; however, the N-acetylglucosaminidase specific activity was decreased significantly in the gradient fraction and only 1.3 y0 of the total activity of the homogenate was in this fraction. A mitochondrial fraction was also prepared from slime mold cultures which had been treated with ethidium bromide for 5 days.
As seen in Fig. 1, the succinate dehydrogenase activity of the mitochondria from the ethidium bromide-treated cells also banded in the sucrose gradient coincidentally with the major band of protein.
The buoyant density of the most purified mitochondrial fraction as indicated by the highest activity of succinate dehydrogenase was 1.181 f 0.002 g per ml.
It should be noted that this value is identical to that obtained for purified mitochondria from control cultures of slime mold amoebae. The degree of contamination by other organelles of the most purified mitochondrial fraction from ethidium bromide treated cultures is presented in Table I. The recoveries of succinate dehydrogenase, catalase, and N-acetylglucosaminidase in the gradient fraction of 1.181 buoyant density were, respectively, 24, 4.5, and 1 y0 of the total activity of the homogenate.
Hence, the mitochondrial fraction obtained from ethidium bromidetreated cells has an identical degree of contamination by lysosomes and peroxisomes as does the mitochondrial fraction obtained from control cultures. The 7700 X g pellet is the once washed crude mitochondrial pellet. The fraction from the gradient with the highest succinate dehydrogenase activity (see Fig. 1) was analyzed for catalase and N-acetylglucosaminidase, as described under "Methods." Treatment with ethidium bromide was for 5 days.  The enzymes were assayed as described under "Methods" in whole cells lysed with Triton X-100 or in mitochondria obtained from the most purified fraction of the sucrose gradient.
Each value is the mean f the standard error of the mean of six different experiments. Once the purity of the mitochondrial fractions obtained from both control and ethidium bromide-treated cells had been established and shown to be comparable, the activities of several enzymes and enzyme complexes of the mitochondrial respiratory chain were determined.
As seen in Table II, the specific activities of succinate and NADH dehydrogenases, were significantly higher (144 to 166%) in whole cells of ethidlum bromide-treated as compared to control cultures. IYo significant differences, however, were observed in the specific activity of either enzyme in the purified mitochondrial fraction from control and ethidium bromide-treated cells. The amount of mitochondrial protein present in 100 mg of whole cells was calculated by dividing the total activity of either succinate or NADH dehydrogenase in the cells by the specific activity of the enzyme in the most purified gradient fraction.
The mitochondrial protein content in control cells was determined to be 21.1 mg/lOO mg of cell protein using succinate dehydrogenase and 22.6 mg/lOO mg of cell protein using NADH dehydrogenase. By contrast, the mitochondrial protein content of the ethidium bromide-treated cells was 31.5 and 30.5 mg/lOO mg of cell protein calculated using these enzymes.
Previously (10) we reported that the addition of ethidium bromide to slime mold cultures immediately blocked the formation of cytochromcs a-~, determined either spectrophotometrically or enzymatically.
The synthesis of cytochrome b, determined either spectrophotometrically or enzymatically, using succinate-cytochrome c reductase, was also inhibited by ethidium bromide treatment but at a lower rate. The activity of these enzyme complexes was compared in purified mitochondria obtained from both control and ethidium bromide-treated cells (Table 11). The specific activity of cytochrome oxidase was decreased 807,, while the specific activity of succinate-cytochrome c reductase was decreased nearly 50%.
Spectral analyses of the purified mitochondria from control and ethidium bromide-treated cells confirmed the decreases in enzymatic activity of these two segments of the respiratory chain. The characteristic bands of cytochromes u-u3, b, and c-cl were observed at 605, 562, and 552 nm, respectively, in the purified mitochondria (Fig. 3). The total amounts of cytochromes a-a3 and b were decreased 82yo and 35%, while that of c-c1 was increased 21 y0 after treatment with ethidium bromide (Table III) more apparent when the dithionite-reduced mitochondria were analyzed using ascorbate-tetramethylphenylenediamine reduced mitochondria in the reference cuvette (Fig. 4, bottom panel). Tzagoloff et al. (19) have established that formation of the membrane-bound oligomycinsensitive ATPase in yeast mitochondria requires proteins synthesized within the mitochondria. These hydrophobic proteins appear to be present in the membrane and necessary for the attachment of the ATPase (Fl) to the membrane.
To determine whether ethidium bromide treatment of slime mold cultures had any effect on the formation of the oligomycin-sensitive ATPase, it was first necessary to establish optimal conditions for assaying the enzyme. Fig. 4 indicates that the optimal rate of the Mg%timulated ATPase in purified

+21%
ATPase was assayed as described under "Methods" using the most purified mitochondrial fraction from the gradient. Where indicated 10 rg of oligomycin were added. Treatment with ethidium bromide was for 5 days.
slime mold mitochondria was observed at pH 9.0. Oligomycin, added at a final concentration of 10 pg per ml, also had maximum inhibitory effects when the enzyme was assayed at pH 9.0. In separate experiments (data not shown), it was determined that maximum inhibition was obtained at oligomycin concentrations of 5 w: per ml when 0.5 to 1 mg of protein was used in the assay. The addition of the uncoupler, dinitrophenol, did not cause any stimulation of ATPase activity in purified slime mold mitochondria.
The total ATPase activity of purified mitochondria from ethidium bromide-treated cells is decreased 66% compared to mitochondria obtained from control cells (Table IV)