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The rod outer segment disk membrane of bovine retina has been isolated in a predominantly fused state. The physical and chemical properties of the membrane in the fused state are profoundly different from the corresponding properties of the same membrane in the unfused state. Exposure to light induces the transition of the disk membrane from the fused to the unfused state. Evidence is presented which suggests that the fusion-defusion cycle of the disk membrane is a primary event of photoexcitation and nerve stimulation.

Two reports in the literature that mitochondria isolated in special media or from a particular source do not undergo configurational changes under energizing, conditions have been analyzed in detail. It could be shown that the failure to demonstrate configurational changes was a consequence of a procedure which allowed anaerobiosis to set in before the mitochondria were fixed. When fixation was achieved while the mitochondria were still under energizing conditions, the correlation between configurational change and change in the energy state could be confirmed.

Ordered arrays of structured material were visualized in the intracristal space of isolated beef heart mitochondria in two ways. Under standard conditions of fixation, structured material in the intracristal space appeared as paracrystalline arrays nestled between two apposing membranes. When mitochondria were preincubated with phosphotungstic acid (PTA) prior to fixation, the structures in the mitochondrial intracristal space took on an open lattice structure. Such structures, either paracrystalline or lattice, could not be demonstrated in the mitochondrial matrix space under these conditions.

The energized configuration of the cristal membrane of beef heart mitochondria can be maintained only as long as oxygen is available for electron transfer. When the oxygen supply is exhausted, the membrane undergoes a transition to the nonenergized configuration. Since the exhaustion of the available oxygen supply is complete in 5–20 sec, it is impossible to apply the method of sedimenting the mitochondria prior to fixation for studying the energized configurational states of mitochondria. The direct addition of glutaraldehyde followed by osmium tetroxide to the mitochondrial suspension is the most effective way of freezing the configurational state of the cristal membrane. Fixation with glutaraldehyde appears to be complete within 1–2 sec even at 0°. Osmium tetroxide alone can also “freeze” the energized configuration by fixation but the concentration of the fixative is critical. The problem of capturing the configurational state applies not only to energized transitions (nonenergized to energized) but also to nonenergized transitions (orthodox to aggregated). The freezing by fixation of the cristal membrane in the aggregated configuration is best accomplished by the sequential use of glutaraldehyde and osmium tetroxide. When the levels of glutaraldehyde and osmium tetroxide are respectively too low or too high, the mitochondrion will undergo a transition from the aggregated to the orthodox configuration before fixation is complete. Light-scattering studies provide an independent method for monitoring configurational changes in mitochondria; these light-scattering measurements confirm that the conditions for fixation which lead to stabilization of the energized state as judged by electron microscopy, also show maintenance of configuration as judged by absence of light-scattering changes after the fixatives are introduced. Reagents used in negative staining will induce the geometrical form of the energized configuration of the mitochondrion even under nonenergizing conditions. These reagents are thus unsuitable for use in studies of configurational transitions in mitochondria.

When the cristae of adrenal cortex mitochondria are stabilized in the orthodox configuration by the binding of 20–25 mµmoles/mg protein of either Ca2+ or free fatty acids (oleic acid), both the capacity for carrying out coupled reactions and the capacity for undergoing energized configurational transitions are lost. The coupled reactions studied included ATP synthesis, divalent cation translocation, monovalent cation trnaslocation, and reversed electron transfer. The coupled processes and energized configurational changes are fully operative when the cristae of adrenal cortex mitochondria are in the aggregated configuration. However, two processes that have been shown to depend on conformational changes (the anaerobic-aerobic proton ejection and energized accumulation of inorganic phosphate) still proceed when mitochondria are in the orthodox configuration. When the mitochondria are initially in the orthodox configuration, addition of divalent cations (Mg2+ or Mn2+) or albumin induces a transition of the cristae to the aggregated configuration and leads to restoration of all the coupled processes. the orthodox to aggregated transition is reversible and the modulation of this reversibility appears to be one of the key points of control in the mitochondrion and possibly of cellular functions.

Bovine adrenal cortex mitochondria examined by electron microscopyin situ orin vitro in 0·25 M sucrose have an unusual cristal membrane structure. The cristae usually appear as unconnected vesicles within a double membrane system. A few of the vesicles appear to be attached to the inner boundary membrane or to one or more other vesicles. The configuration of such mitochondria will be defined as the orthodox configuration. In this communication we will provide evidence that the inner membrane is not composed of multiple vesicles, but is one continuous membrane with tubular invaginations, and that these invaginations alternately are ballooned out and squeezed down. A mechanism has been proposed to account for the differentiated structure of the cristae of adrenal cortex mitochondria.

Bovine adrenal cortex mitochondria when isolated in a medium 0·25 M in sucrose contaminated (with calcium) have tubular cristae which are periodically expanded to spherical vesicles and contracted to flattened connecting sections. This scalloped tubular form of the cristae corresponds to the nonenergized orthodox configuration—a configuration in which the matrix space is maximally expanded. When adrenal cortex mitochondria are isolated in media in which the free calcium content is relatively low, e.g., a medium 0·25 M in sucrose and 0·1 mM in EDTA, the cristae assume the aggregated configuration—a nonenergized configuration in which the matrix space is maximally contracted. The composition of the isolation medium determined the configuration. Procedures have been described for isolating bovine adrenal cortex mitochondria (a) predominantly in the orthodox configuration, (b) predominantly in the aggregated configuration, or (c) in a mixed (1:1) population of configurations. The concentration of Ca2+ bound to the mitochondrion was found to be a determinant of the nonenergized configuration. When the level of bound Ca++ was 20–25 µmoles/mg protein, the cristae of the mitochondria were entirely in the orthodox configuration. Addition of Ca2+ could induce the transition of cristae from the aggregated to the orthodox configuration whereas addition of Mg2+ could induce the transition of cristae from the orthodox to the aggregated configuration. The configurational transition could be followed by any of several methods—a change in 90° light scattering, a change in O.D.520 mµ, a change in pH, or examination by electron microscopy. The orthodox to aggregated transition is energy-independent since it proceeds even in presence of inhibitors both of electron transfer and of ATP hydrolysis. The binding of Ca2+ is independent of the binding of Mg2+; this independent binding is consistent with the opposite effects induced by Ca2+ and Mg2+, respectively. Whereas Ca2+ induces a proton release, a decrease in 90° light scattering and a decrease in O.D.520 mµ (when the cristae are initially in the aggregated configuration), Mg2+ induces equal and opposite changes (when the cristae are initially in the orthodox configuration).