Abstract
The objective of this investigation was to determine if a mitochondrial inner membrane protein coded for by the nuclear genome could be identified with the rough endoplasmic reticulum associated with mitochondria. Rough endoplasmic reticulum-mitochondrial complexes were collected by zonal centrifugation. These associations were then subjected to hypotonic-hypertonic treatment and the mitoplasts were separated from rough endoplasmic reticulum and outer membranes. These were analysed for various marker enzymes including succinic dehydrogenase, succinate cytochrome c reductase, and cytochrome oxidase for inner mitochondrial membrane, monoamine oxidase for outer mitochondrial membrane, and glucose-6-phosphatase for rough endoplasmic reticulum. Some inner membrane damage occurred and contaminated the RER fraction as determined by cytochrome oxidase activity. However, the ratios of monoamine oxidase: cytochrome oxidase and glucose-6-phosphatase: cytochrome oxidase were over 20-fold and 105-fold greater, respectively, than for intact mitochondria indicating that inner mitochondrial membrane contamination was very low. Interestingly, succinic dehydrogenase activity was found to be present in the rough endoplasmic reticulum-outer mitochondrial membrane fraction, but succinate cytochrome c reductase activity was not detectable. In addition, the ratio of succinic dehydrogenase: cytochrome oxidase activity was 9-fold higher in the RER fraction than in mitochondria. These results were interpreted to mean that the succinic dehydrogenase activity in this fraction did not represent an enzyme which was a component of the electron transport chain. Specific activity of succinic dehydrogenase in the rough endoplasmic reticulum-outer mitochondrial membrane fraction was diminished in material from fasted rats and was increased in fasted-refed animals. Succinic dehydrogenase was also found localized on the rough endoplasmic reticulum by electron microscopic procedures using tetrazolium salts. These experiments support the hypothesis that succinic dehydrogenase is synthesized on the mitochondrially associated RER in preparation for transfer to the mitochondria.
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