By means of a new "quick-sampling" method, micropellets of mouse liver mitochondria were rapidly prepared for electron microscopy during the recording of steady state metabolism. Reversible ultrastructural changes were found to accompany change in metabolic steady states. The most dramatic reversible ultrastructural change occurs when ADP is added to systems in which only phosphate acceptor is deficient, i.e., during the State IV to State III transition as defined by Chance and Williams. After 15 min in State IV, mitochondria display an "orthodox" ultrastructural appearance as is usually observed after fixation within intact tissue. On transition to State III, a dramatic change in the manner of folding of the inner membrane takes place. In addition, the electron opacity of the matrix increases as the volume of the matrix decreases, but total mitochondrial volume does not appear to change during this transition. This conformation is called "condensed." Isolated mitochondria were found to oscillate between the orthodox and condensed conformations during reversible transitions between State III and State IV. Various significant ultrastructural changes in mitochondria also occur during transitions in other functional states, e.g., when substrate or substrate and acceptor is made limiting. Internal structural flexibility is discussed with respect to structural and functional integrity of isolated mitochondria. Reversible changes in the manner of folding of the inner membrane and in the manner of packing of small granules in the matrix as respiration is activated by ADP represent an ultrastructural basis for metabolically linked mechanical activity in tightly coupled mitochondria.

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