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FACTORS INFLUENCING THE DETERMINATION OF THE INTRACELLULAR DISTRIBUTION OF ISOCITRIC DEHYDROGENASE IN RABBIT CEREBRAL CORTEX

JAMES A. SHEPHERD 1

1 Department of Neurology, General Hospital, State University of Iowa, Iowa City, Iowa

Washing the sediment or the mitochondrial fraction of rabbit cerebral cortex homogenates in the process of tissue fractionation increases its isocitric dehydrogenase activity thus giving anomalous recovery results. This increase on washing is not due to the removal of a soluble inhibitor of the dehydrogenase itself.

Isocitric dehydrogenase activity is associated with the mitochondrial fraction and soluble fraction of rabbit cerebral cortex homogenates. It is unlikely that the mitochondrial or soluble activities have been derived from one another, or are due entirely to contamination of one fraction with another. The enzymes themselves from these sources have not been shown to be either the same or different enzymes. The activation of the particulate activity by freezing and thawing has been used as a roughly quantitative test for particulate bound activity. Mitochondrial bound enzyme may be activated by freezing and thawing, exposure to sonic vibrations, the shearing effect of Waring Blendor blades, and partially by washing. The latter may be explained on the basis of permeability factors aggravated by removal of isocitrate within the mitochondria by aconitase. Rabbit cerebral cortex mitochondria are not easily ruptured by homogenization in the Potter and Elvehjem homogenizer in sucrose suspension. The increase in specific activity of the nuclear fraction due to freezing and thawing is larger than that of the mitochondrial fraction, though the actual increase in activity is much smaller. The presence of a nuclear isocitric dehydrogenase has not been ruled out. A highly active isocitric dehydrogenase in soluble form has been prepared from rabbit cerebral cortex mitochondria.

Submitted on June 13, 1955


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