Intracellular Localization of Enzymes in Spleen

Abstract

In 1949, Hogeboom (1) reported that in rat liver homogenates which were prepared in 0.88 M sucrose and separated into four fractions by differential centrifugation, 32 and 58 per cent of the total D P N H 1 cytochrome c reductase activity of the original homogenates were recovered in the mitochondria and microsomes, respectively. He also found that, based on activity per mg. of nitrogen, the enzyme was concentrated 3.3 times in the microsome fraction and only 1.3 times in the mitochondria with respect to the homogenate. In 1951, as part of a study of the D P N H cytochrome c reductase of rabbit liver and heart mitochondria, Eichel (2) observed a 1.6-and 2.9-fold concentration of the enzyme in mitochondrial fractions prepared from isotonic sucrose homogenates of liver and heart, respectively. Cytochrome c oxidase assays on these (and other) fractions and the original homogenates confirmed the mitochondrial nature of the isolated fractions. Subsequently, Brody et al. (3) studied the distribution of D P N H cytochrome c reductase in various fractions obtained from homogenates of rat brain cerebral cortex. In contrast to the results with rat liver, they found a 2.8-fold concentration of the enzyme in the mitochondria with respect to the original homogenate, while the specific activity of the microsomal fraction was slightly less than that of the original homogenate. No data were given for the recovery of total enzyme activity in each of the particulate fractions. Strittmatter and Ball (4) and de Duve el al. (5) have also studied the distribution of the reductase in rat liver fractions. Their results are in essential agreement with those reported previously.

Intracellular Localization of Enzymes in Spleen

“…These figures are in excellent agreement with those obtained in liver. The presence of more than twice as much R N A in the nuclear fraction of spleen as in the corresponding fraction of liver parallels previous findings in the nuclear fractions of these two tissues with respect to N content and D P N H cytochrome c reductase activity (1). Total reductase and total N recovered in spleen nuclei were greater than those found in liver nuclei by a R N A -P data listed by Leslie (24).…”

“…Finally, it is of interest to compare the RNase distribution with the results previously reported on the localization of some oxidative enzymes in spleen (1). Cytochrome c oxidase and succinic cytochrome c reductase were localized almost exclusively in the mitochondria, while DPNH cytochrome c reductase was split between the mitochondria and microsomes.…”

“…factor of at least 2. It is probable, as has been contended elsewhere in regard to the localization of respiratory enzymes in spleen (1), that a large part of the R N A recovered in spleen nuclear fractions is due to contamination with unbroken cells and possibly adsorbed microsomes. This idea is supported by the results in Table I, which show that the spleen microsomal fraction is the only one in which R N A is concentrated in terms of total N, the average R N A / N ratio being 692/1, as compared with 278/1 for the whole homogenate.…”

“…Homogenization of the spleens and separation of the four subcellular fractions were carried out according to the procedures previously described (1). Male Wistar rats (Charles River Labs, Brookline, Massachusetts), weighing 200 to 400 gm and fed Purina fox chow checkers, were used, except where indicated otherwise.…”