INTRACELLULAR LOCALIZATION OF ENZYMES IN SPLEEN

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Abstract
The intracellular distribution of nitrogen, reduced diphosphopyridine nucleotide (DPNH) cytochrome c reductase, succinic dehydrogenase, and cytochrome c oxidase was studied in fractions derived by differential centrifugation from rat and guinea pig spleen homogenates. In each species, the nuclear fraction accounted for 40 to 50 % of the total N content of the homogenate, and the mitochondrial, microsome, and supernatant fractions contained about 8, 12, and 30 % of the total N, respectively. Per mg of N, DPNH cytochrome c reductase was concentrated in the mitochondria and microsomes of both rat and guinea pig spleens. Seventy % of the total DPNH cytochrome c reductase activity was recovered in these 2 fractions. The reductase activity associated with the nuclear fraction was lowered markedly by isolating nuclei from rat spleens with the sucrose-CaCl2 layering technique. The lowered activity was accompanied by the recovery of about 90 % of the homogenate DNA in the isolated nuclei, indicating that little, if any, of the reductase is present in spleen cell nuclei. Per mg of N, succinic dehydrogenase was concentrated about 10-fold in the mitochondria of rat spleen, and 65 % of the total activity was recovered in this fraction. Cytochrome c oxidase was concentrated, per mg of N, in the mitochondria of both rat and guinea pig spleens. The activity associated with the nuclear fraction was greatly diminished when this fraction was isolated from rat spleens by the sucrose-CaCl2 layering technique. Only 50 to 70 % of the total cytochrome c oxidase activity of the original homogenates was recovered among the 4 fractions from both rat and guinea pig spleens, while the specific activities of reconstructed homogenates were only 55 to 75 % of those of the original whole homogenates. This was in contrast to the results with DPNH cytochrome c reductase and succinic dehydrogenase, where the recovery of total enzyme activity approached 100 %, and the specific activities of reconstructed homogenates equalled those of the original homogenates. The recovery of cytochrome c oxidase was greatly improved when only the nuclei were separated from rat spleen homogenates. Data were presented comparing the concentrations (ratio of activity per mg of N of the fraction to activity per mg of N of the homogenate) of DPNH cytochrome c reductase in mitochondria and microsomes derived from different organs of different animals. Data were presented comparing the activities per mg of N of DPNH cytochrome c reductase in homogenates from several organs of various animals.