Thesis

Mitochondrial enzyme activity during the cell cycle of Polytomella agilis

A population of Polytomella agilis was synchronized with hydroxyurea, which inhibits cells at the onset of DNA synthesis (the G1/S border). To gain information on the sites of mitochondrial enzyme synthesis (succinic dehydrogenase, NADH dehydrogenase and cytochrome c oxidase} or proteins involved in their assembly or incorporation into membranes, the effects of chloramphenicol (an inhibitor of mitochondrial protein synthesis) and cycloheximide, an inhibitor of extramitochondrial protein synthesis) on the specific activities of these enzymes were studied, through the cell cycle. In untreated cells, peak patterns of enzyme synthesis were observed. A mitochondrial site of succinic dehydrogenase is suggested since its activity declined in chloramphenicol-treated cells during the first cycle. Cycloheximide had no effect on its activity. NADH dehydrogenase is apparently synthesized under mitochondrial control since chloramphenicol results in decreased activity in the first cycle. In the subsequent cell cycle little, if any, activity was detected. Cycloheximide affected the pattern of activity during the cell cycle, suggesting the inhibition of some cytoplasmic components involved in the regulation of the assembly, membrane-integration or activity of the enzyme. In the presence of chloramphenicol, cytochrome c oxidase activity did not decline until the second cell cycle. Enzyme activity was not inhibited in either cycle by cycloheximide. This suggests a mitochondrial site of synthesis. The diminished activities of both cytochrome c oxidase and NADH dehydrogenase in the second cycle following the addition of chloramphenicol suggests that the mitochondrion of P. agilis arises by growth and division. Cell division was not inhibited by chloramphenicol after the first cell cycle, suggesting that a completely functional mitochondrion is not an absolute requirement for cell division. Cell division does depend on cytoribosomal protein synthesis, since it is blocked by cycloheximide.

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