Observations on the fragmentation of isolated flight-muscle mitochondria from Calliphora erythrocephala (Diptera)

Abstract

A combined electron-microscopic and biochemical study has been made of the disintegration of isolated mitochondria from asynchronous flight muscle of the blowfly Calliphora erythrocephala . These mitochondria are particularly suitable because they can be easily isolated free from other intracellular structures and because of the regularity of their closely packed cristae, seen in fixed (sectioned) and unfixed, negatively stained material. Fragmentation occurs on treatm ent with distilled water, or more rapidly and extensively on sonication. The first stage detected in the disruption of the plate-like cristae is the formation of tubular elements, observed in sectioned preparations. These were seen after negative staining as irregular ribbons, studded with the stalked particles, 80 to 95 Å in diameter, first described by Fernández-Morán. Further fragmentation of the tubules yielded rounded fragments, about 200 to 2000 Å in diameter, apparently by transverse splitting and lateral ‘budding’. These were vesicles, as shown in sectioned material, bearing stalked particles seen by negative staining. After removal of the larger fragments by centrifugation, sonicated material was fractionated on a sucrose density gradient. Protein, cytochrome oxidase activity and rate of reduction of ferricyanide by succinate were determined. A middle fraction with the highest enzymatic activity/ml. of sucrose was found to be a concentrated suspension of vesicles, 200 to 500 Å in diameter, still bearing stalked particles. Little recognizable material was seen in a fraction near the top of the gradient with minimal activity and maximal concentration of (‘soluble’) protein. Available evidence indicates that the cytochrome oxidase and succinate-ferricyanide activities become associated with the vesicles. The activity/mg of protein of the most active fraction was not more than double that of the sonicate. The smaller fragments seemed to be more deficient in cytochrome c than the larger. The stalked particles were firmly attached to cristae, tubules and vesicles, and attempts to remove them were unsuccessful.