Abstract
Mitochondria were isolated from the cellular slime mold. Dictyoostelium 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 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 a-a3 was decreased over 80% and that of cytochrome oxidase and oligomycin sensitive ATPase were reduced approximately 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 (Stuchell, R. N., Weinstein, B. I., and Beattie, D. S. (1973) Fed. Eur. Biochem. Coc Lett. 37, 23-26). 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. Consequently, the amount of mitochondria, or mitochondrial protein per cell, calculated from the activity of NADH and succinate dehydrogenases has increased nearly 50%. Possible models to explain the control of mitochondrial biogenesis are discussed to explain these results.
Highlights
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 minor band of protein containing succinate dehydrogenase activity was observed near the top of the gradient, possibly due to fragments of mitochondrial membranes damaged during homogenization of the cells
It should be noted that the formation of NADH and succinate dehydrogenases has been shown torequire proteins synthesized only on cycloheximide-sensitive cytoplasmic ribosomes [13, 20]
Summary
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. 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 specific activities of NADH and succinate dehydrogenases were identical in the purified mitochondria from control and ethidium bromide-treated cells. Treatment of human fibroblast cultures [5], L cells [6, 7] and SV40-transformed cells [8] with ethidium bromide for several days caused a drastic reduction in the cytochrome u-u3 content and a variable decrease in the level of cytochrome b. The cellular slime mold Dictyostelium discoideum is an excellent organism for studies of both mitochondrial biogenesis and the role of mitochondria during cellular differentiation
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