Tissue fractionation studies. 2. The nature of the linkage between acid phosphatase and mitochondria in rat-liver tissue

Abstract

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

Tissue fractionation studies. 9. Enzymic release of bound hydrolases

“…For the measurement of free activities, the substrate mixtures were made up so as to realize the assay conditions described by and by de Duve, Pressman, Gianetto, Wattiaux & Appelmans (1955); they contained in addition all the components of low molecular weight present in the pre-incubation medium. As observed before (Berthet, Berthet, Appelmans & de Duve, 1951) and again here in some preliminary experiments, lack of the last-named precaution may be responsible for the osmotic rupture of the particles, if they are permeable to one or more of the omitted components. The methods used for stopping the reactions and performing the subsequent analyses were those described previously .…”

“…Such a role has been suspected before on the basis of temperature and pH effects (Berthet et al 1951; Table 2. Influence of lecithina8e on 8olubility of ly8osomal hydrola8e8 A mitochondrial preparation was pre-incubated for 10 min.…”

“…Owing to adsorption phenomena, the values sly related, but for physical release (solubilization) are underiince the former estimated; on the other hand, the accessibility of te, whereas the the enzymes (free activities) may be overestimated ily those of the if further disruption of the particles occurs during )f the barrier in the assays themselves. With acid phosphatase, depend only on which is known to be fairly strongly adsorbed by )e influenced by mitochondrial sediments (Berthet et al 1951), 3nzymes and of solubilization may be considered as practically complete and suffices therefore to account for the he differences in activation results. This is also true of P-glucuronidwhich were oc-ase, but not of the acid nucleases, a fair proporesent investiga-tion of which appears to remain attached to close parallelism particles.…”

Histamine Release From Isolated Rat Mast Cells in Metal Ion-Free Medium

“…Favoring this view is the fact that the presence of sucrose decreases additional histamine release by NaCI from the mast cell precipitates (3,000g) after application of compound 48/80. These results and the observation of Berthet and others (14) that the release of enzyme by NaCI from liver lysosomes was inhibited by sucrose, indicate that sucrose prevents the release of various substances from subcellular granules, although this mechanism remains unexplained.…”

CONCENTRATION OF ACID PHOSPHATASE, RIBONUCLEASE, DESOXYRIBONUCLEASE, SS-Glucuronidase, AND CATHEPSIN IN "DROPLETS" ISOLATED FROM THE KIDNEY CELLS OF NORMAL RATS

“…It has been suggested (1) that these granules may be related to "secretory granules" and "Golgi bodies" and to fractions rich in add phosphatase which had been isolated from the cells of the liver of the rat by Palade (3), Novikoff, Podber, Ryan, and Noe (4), and Berthet el al. (5). De Duve et al (6) have shown recently that this liver fraction is distinct from the mitochondrial fraction and that acid phosphatase, ~-glucuronidase, ribonuclease, desoxyribonudease, and cathepsin are concentrated in it.…”