Tissue fractionation studies. 1. The existence of a mitochondria-linked, enzymically inactive form of acid phosphatase in rat-liver tissue

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Abstract
In fresh rat-liver homogenates prepared in 025 [image]sucrose, a large proportion is linked with the mitochondria. The bound enzyme had little or no activity towards added glycerophosphate at pH 5. Ageing of the prepns. at 0s, short exposure to the Waring blendor, repeated freezing and thawing, all lead to an irreversible release of the enzyme, which then appeared to a large extent in a soluble form. Partial damage to the complex was caused by the procedures used to homogenize and fractionate liver tissue. In contrast to its bound form, the free enzyme exhibited a high activity. When mitochondria were separated by differential centrifugation and washed once, they were found to contain 55-60% of the total acid phosphatase content of the original homogenate. The remainder was found partly in the smaller granules, partly in the final supernatant. The nuclear fraction contained only a low percentage of the total activity. Analysis of these results indicated: (a) that the whole acid phosphatase content of rat liver must be particle-bound in the intact tissue; (b) that the ability to bind acid phosphatase in the manner described belongs to a population of granules which are fairly heterogeneous in size. About 1/3 them were too small to sediment completely in 10 min. at 8500 g. The implications of these findings, in particular with respect to the technique of centrifugal fractionation, are discussed.