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In the preceding paper , the existence in rat-liver tissue of a mitochondrialinked form of acid phosphatase has been described and the cytological significance of this finding has been discussed. The nature of the linkage between enzyme and granule forms the object of the present communication.Two facts relevant to this problem have already been reported, namely the great lability of the complex, which can be disrupted quantitatively by mechanical or physical means (Waring blender, freezing and thawing, hypotonic media), and its lack of enzymic activity towards added glycerophosphate at pH 5. Considerations based on these and other properties to be described in this paper, indicate that the enzyme is retained inside the mitochondrial structure by a membrane-like barrier, endowed with selective permeability and impermeable to glycerophosphate. A preliminary report of this work has already been published (Duve, Berthet, Berthet & Appelmans, 1951). METHODSThe following general procedure has been followed in many of the experiments which will be reported. Mitochondria were separatedfromrat-liver tissue bythe method previously described (Berthet &8 Duve, 1951), washed once and resuspendedin a small quantity ofcold 0-25M-sucrose. Samples, of this suspension were suitably diluted with media of given composition and incubated. Except in the cases where the effect of temperature was studied, the incubation was carried out in the cold room, in an ice-water bath.At various times after the beginning of the experiment, samples of the diluted suspension were centrifuged 10 mis. at 20,000 g. Part of the supernatant was collected and kept for enzyme assay. A sample for the determination of the starting value was obtained in a similar way from a 0-25Msucrose dilution ofthe mitochondria. Another portion of the mitochondria was treated for 3 min. in the Waring blender, for the measurement of the total enzyme content.When all the samples had been collected, their acid phosphatase activity was determined according to the technique described previously to provide an index of the amount of soluble enzyme present at the time the suspension was centrifuged.In a number ofexperiments, additions to this procedure or deviations from it were introduced; these are mentioned in the text. RESULTSEffect of temperature. When mitochondria are kept in 0-25M-sucrose at 00, acid phosphatase is released very slowly, as shown by many of the graphs reproduced below. Raising the temperature produces a marked increase in the rate of liberation of the enzyme, and the activation which occurs under those conditions follows' a characteristic course. The results which are represented graphically in Figs. 1-3 illustrate various aspects of this phenomenon. 0 o to cut X~> 50 D 25 co 0 1 2 3 4 Time (hr.) Fig. 1. Influence of temperature on release of acid phosphatase in 0 25M-sucrose. 2 0 100 -o 0co 1UL 75 --°380 50 _ 380 .0 380 00 25 -3

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