The cytochrome oxidase content of rat tissues was investigated, utilizing the accumulation of the free radical formed by the emzymatic, univalent oxidation of a stable, non-toxic substrate, N-phenyl- p-phenylenediamine ( p-aminodiphenylamine). This procedure eliminated the non-enzymatic coupling of the radical with α-naphthol, found in the classic Nadi reaction. Using homogenates, the following decreasing order of enzyme activity was found: heart, kidney, diaphragm, liver, rectus abdominis. A more sensitive assay was also developed, involving the reduction of cytochrome c by ascorbic acid, and using 2-( p-iodophenyl)-3-( p-nitrophenyl)-5-phenyl tetrazolium chloride to analyze unoxidized ascorbic acid. The same order of tissue activity was demonstrated. The cytochrome oxidase content of a tissue may be a reflection of its physiological role.

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1. The ability of tricarboxylic acid-cycle metabolites to influence the physiological performance of the perfused anaerobic rat heart was investigated. Energy expenditure/h [(beats/min)x60xsystolic pressure/g of protein] for various anoxic conditions compared with oxygenated control hearts were: 5mm-glucose, 4.5%; 20mm- or 40mm-glucose, 10%; 20mm-glucose plus fumerate+malate+glutamate, 29%; 20mm-glucose plus oxaloacetate and alpha-oxoglutarate, 31%. 2. The energy expenditure/lactate production ratio was increased by the tricarboxylic acid-cycle metabolites, indicating that alterations in anaerobic physiological performance did not result from changes in glycolysis. 3. Analysis of tissue constituents provided further indication of an enhanced energy status for fumarate+malate+glutamate- and oxaloacetate+alpha-oxoglutarate-perfused hearts; tissue concentrations of both glycogen and ATP were higher than in the 20mm-glucose-perfused groups. 4. A marked increase in the accumulation of succinate in tissues perfused with oxaloacetate+alpha-oxoglutarate or fumarate+malate+glutamate provided further evidence that these metabolites were stimulating mitochondrial energy production under anoxia. 5. These studies indicate that mitochondrial ATP production can be stimulated in an isolated mammalian tissue perfused under anaerobiosis with a resulting enhancement of cell function.

Mitochondria were isolated from rat liver homogenate by both zonal and sedimentation equilibrium centrifugation. From electron microscopic examination of thin sections it was observed that 81% of the isolated mitochondria were in contact with rough endoplasmic reticulum (RER). Intact, non-sectioned mitochondria subjected to negative staining procedures appeared to show points of connection between RER and the outer mitochondrial membrane. After treatment of mitochondria with digitonin to remove outer membranes, some of the resulting mitoplasts (intact inner membranes) remained closely associated with RER. Serial section analysis of intact rat liver indicated that RER saccules fit over mitochondria like caps providing broad areas of contact between the two organelles. The RER saccules were also observed communicating with more than one mitochondrion.