R. Wattiaux

Nucleosidediphosphatase activity in plasma membrane of rat liver.

Abstract 1. 1. The intracellular distribution of nucleosidediphosphatase activity has been investigated in rat liver with ADP and IDP as substrates and compared with those of reference enzymes. 2. 2. After differential centrifugation in 0.25 M sucrose, the distribution of the enzyme acting on IDP is similar to that of glucose-6-phosphatase; ADPase distribution is comparable to that of 5′-AMPase. 3. 3. Deoxycholate considerably increases nucleosidephosphatase activity on IDP, but has little effect when ADP is used as the substrate. The detergent does not affect the distribution of ADPase; on the contrary, significant changes of the IDPase distribution are observed if the test is performed with or without deoxycholate. 4. 4. Enzyme distribution curves have been determined after centrifugation in a sucrose gradient of a particle preparation obtained by centrifuging the homogenate at 3.7·10 6 × g· min . When granules are layered at the top of the gradient no clear distinction is seen between the enzyme distribution curves. When granules are deposited at the bottom of the gradient, a striking separation of the nucleosidediphosphatase distribution curves can be observed. The behaviour of IDPase is similar to that of glucose-6-phosphatase; ADPase behaves like 5′-AMPase. 5. 5. In purified plasma membrane preparations, ADPase and 5′-AMPase show a specific activity approx. 25 times higher than that fund in the homogenate. IDPase is only 5 times purer than in the homogenate. Some properties of the nucleosidediphosphatase associated with the plasma membrane are given. 6. 6. The results are discussed with respect to the presence in rat liver of two nucleosidediphosphatases, one associated with the plasma membrane and the other with endoplasmic-reticulum membranes.

Lysosomes in rat-kidney tissue

Summary 1. The intracellular distribution of catalase and several acid hydrolases has been investigated on homogenates of rat kidney and compared with those of cytochrome oxidase (EC 1.9.3.1), glutamic dehydrogenase (EC 1.4.1.2) and glucose-6-phosphatase (EC 3.1.3.9), used as reference enzymes for mitochondria and microsomes. 2. Acid hydrolases and catalase are principally associated with fractions sedimenting at low centrifugal forces, but they also show significant activities in the soluble fraction. Their distribution patterns are quite different from those of cytochrome oxidase, glutamic dehydrogenase and glucose-6-phosphatase. 3. Distribution curves of acid hydrolases, catalase (EC 1.11.1.6) and two mitochondrial enzymes, cytochrome oxidase and glutamic dehydrogenase, have been determined after density equilibration in a sucrose gradient of a total mitochondrial fraction. Acid hydrolases show flattened distribution curves in contrast to the remarkably homogeneous distribution of mitochondrial enzymes. Catalase is found in the higher density regions of the gradient. 4. The possible role of these two enzymes in insect flight metabolism is discussed. 5. Acid hydrolases and catalase show the phenomenon of structure-linked latency but hypotonic treatment does not greatly change the activity of the latter enzyme. 6. The injection of Triton WR1339, which lowers the density equilibration of lysosomal enzymes in the liver, has a similar effect on kidney acid hydrolases. However, the action of this substance is not so marked as for the liver enzymes. Catalase and glutamic dehydrogenase do not seem to be affected by this treatment. 7. These results are discussed with respect to the presence of lysosomes in the rat kidney and to their properties.

INFLUENCE OF THE INJECTION OF A SUCROSE SOLUTION ON THE PROPERTIES OF RAT-LIVER LYSOSOMES.

IN work recorded here, rats were injected intraperitoneally with 8 ml. of a 50 per cent sucrose solution. They were killed 2 h after the injection. Some of the properties of the hepatic lysosomes were found to be deeply affected by the treatment, namely: (1) their density was reduced, as shown by a shift towards the regions of low density in the distribution curves of their hydrolases, following density equilibration in a glycogen gradient (Fig. 1); (2) their size was increased, as suggested by a fall of the sedimentation boundaries of their enzymes in the centrifuge tube, after incomplete sedimentation in a stabilizing gradient of aqueous sucrose (Fig. 2). On the other hand, the distribution of proteins, used as marker for mitochondria1 and of particle-bound catalase, remained practically unaltered.

Distribution of mitochondrial enzymes after isopycnic centrifugation of a rat liver mitochondrial fraction in a sucrose gradient: Influence of the speed of centrifugation

Abstract The distributions of mitochondrial enzymes after isopycnic centrifugation of a rat liver mitochondrial fraction in a sucrose gradient differ depending on whether centrifugation is performed at 39,000 rpm or 50,000 rpm in the Spinco SW50.1 rotor. This finding is best explained by postulating that mitochondria are damaged when centrifugation speed is too high.

Influence of the injection of "Triton WR-1339" on lysosomes of a rat transplantable hepatoma.

LYSOSOMES are subcellular particles in which several acid hydrolases of various specificities are localized. One of their main intracellular functions is to digest exogenous compounds taken up by the cell. The lysosomal system can be overloaded with substances which cannot be degraded, at least at a sufficient rate, by hydrolases1. This occurs in rat liver lysosomes2 and to some extent in kidney3 and spleen4 lysosomes after injection of ‘Triton WR-1339’, a non-ionic detergent. This compound is taken up by liver, kidney and spleen cells, and accumulates inside the lysosomes because it is not subjected to the lytic action of lysosomal enzymes. Accumulation causes increase in granular size and decrease in granular density.

Subcellular particles in tumors—III

Abstract 1.(1) The distribution of mitochondrial enzymes has been investigated after isopycnic centrifugation of mitochondrial fractions of HC hepatoma and Morris hepatomas 7794B, 7316A and 16. Two kinds of density gradient were used, one consisting of glycogen dissolved in aqueous sucrose of different molalities and the second of aqueous sucrose alone. 2.(2) In sucrose gradient experiments, the distributions of cytochrome oxidase, monoamine oxidase and glutamate dehydrogenase were established. Isopycnic centrifugation was performed at two speeds, 39,000 rev/min and 65,000 rev/min. At 39,000 rev/min, a similar distribution was seen for the three enzymes in each hepatoma; however, the distribution exhibited marked differences from one hepatoma to the next. The distributions of the three mitochondrial enzymes of hepatomas 16 and HC remained practically unchanged when centrifugation was performed at 65,000 rev/min instead of 39,000 rev/min. In contrast, the higher centrifugation speed did induce changes in the distributions of hepatoma 7316A and 7794B enzymes. 3.(3) In glycogen gradient experiments, cytochrome oxidase distribution was studied. The distribution indicates that the behavior of mitochondria of hepatoma 7794B, 7316A and HC in such gradients is qualitatively similar to that of rat liver mitochondria. However, the parameters that characterize the intramitochondrial spaces, calculated from the distribution pattern, differ to a variable extent from those calculated for rat liver particles. The cytochrome oxidase of hepatoma 16 behaves as if the mitochondria of this tumor were devoid of an osmotic space.

Particules subcellulaires dans les tumeurs

Resume 1.1. Les fractions mitochondriale et microsomiale dun hepatome transplantable chez le rat ont ete analysees par centrifugation isopycnique. Deux sortes de gradients ont ete utilisees: lun de saccharose aqueux pur, lautre de glycogene dissous dans des solutions de concentration variable en saccharose. 2.2. Apres equilibration de densite, dans un gradient de saccharose, dune fraction mitochondriale, la cytochrome oxydase montre une distribution sensiblement homogene, tandis que les hydrolases acides presentent des courbes de distribution plus etalees dans les regions de haute densite du gradient. Apres centrifugation isopycnique dans des gradients de glycogene, les profils de distribution de la cytochrome oxydase sont totalement differents de ceux des hydrolase acides. De plus, les densites dequilibre des mitochondries et des lysosomes, deduites respectivement du comportement de la cytochrome oxydase et des hydrolases acides, sont fort affectees par la concentration en saccharose du milieu, quoique differemment pour les deux especes de particules. 3.3. La centrifugation isopycnique dune fraction microsomiale dans un gradient aqueux de saccharose, et dans des gradients de glycogene, suggere fortement que la glucose-6-phosphatase et la catalase sont associees a des elements distincts de la fraction microsomiale. 4.4. La comparaison de nos resultats avec ceux obtenus par de Duve et ses collaborateurs sur le foie de rat [2, 5, 6] suggere que des differences significatives existent entre les mitochondries de lhepatome HW et les mitochondries de foie de rat, aussi bien quentre les structures portant la catalase dans les deux tissus; tout au contraire, les lysosomes semblent fort comparables dans la tumeur et dans le foie.

Nucleoside diphosphatase in purified preparations of rat liver lysosomes

When performing their heterophagic function, the lysosomes and the endocytosis vacuoles have to fuse in order to allow contact between the acid hydrolases and the endocytosed compounds [l] . Such a process suggests some structural similarity between the lysosome membrane and the plasma membrane from which the endocytosis vesicle membrane is made. Several enzymes are known to be linked to the plasma membrane; thus one possible way of comparing the plasma membrane with the lysosome membrane is to study their enzymic content. Recently we have shown that a nucleoside diphosphatase was associated with the liver plasma membrane [2]. The present work indicates that a similar enzyme is present in purified lysosome preparations and is found mainly in the membranous fraction of these preparations.

Intracellular degradation by liver endothelial cells

Investigations were carried out on the intracellular fate of formaldehyde treated bovine serum albumin (F-BSA), in liver non-parenchymal cells. This paper reports the observations and results obtained by us. The first part of our work involved the injecting of the compound into either a) normal rats, b) rats injected with Triton WR 1339 or c) rats treated with mannan. Fractions obtained after differential and isopycnic centrifugation in sucrose gradients, were analysed by SDS-gel electrophoresis and fluorography. The degradation takes place in a two step process. The molecule is first split into radiolabeled compounds that are still acid precipitable. This is followed by the appearance of acid soluble radioactive molecules. In a sucrose gradient the first kind of degradation products exhibit a distribution totally different from that of acid soluble degradation compounds. In the second part of our experiments, fairly pure fractions of the organelles, known to be involved in the endocytic pathway i.e. endosomes, transfer lysosomes and accumulation lysosomes (marked by the presence of either Triton WR 1339 or mannan) were isolated and incubated with [125I]-F-BSA. These experiments revealed that endosomes, isolated by us, are incapable of degradation. Accumulation lysosomes arising exclusively from liver non-parenchymal cells (in which mannan had accumulated) though rich in certain hydrolases eg. arylsulfatase did not have an efficient proteolytic machinery. Our results, both fromin vivo andin vitro studies, suggest that the first degradation step occurs in one type of structure (probably not endosomes), a sort of hybrid endosome-lysosome (as they are not affected by glycyl-l-phenyl-2-napthylamide [1]) and the second step in a different type of lysosomes, what we have designated transfer lysosomes.

Subcellular particles in tumors—IV Lysosomes in hepatoma HC and Morris hepatomas 7794A, 7794B, 5123A, 7316A and 16

Abstract 1. 1 . The activities of seven acid hydrolases were measured in HC hepatoma and in Morris hepatomas 7794 A, 7794 B, 5123 A and 16 . In general the enzymes in the tumors exhibit a specific activity ranging between 50 and 150% of that found in rat liver tissue; however, the specific activities of acid phosphatase, N-acetylglucosaminidase and s-galactosidase in hepatoma 5123 A are far greater than those found in liver homogenates. 2. 2 . The intracellular distribution of these enzymes was established after differential centrifugation in 0·25 M sucrose. The distribution pattern resembles that of the same enzymes in rat-liver homogenates. The enzymes are found mainly in the mitochondrial fractions and exhibit a peak of specific activity in the light mitochondrial fraction. 3. 3 . Total mitochondrial fractions of hepatomas were analyzed by isopycnic centrifugation in a sucrose gradient and in gradients composed of glycogen dissolved in aqueous sucrose of different molalities. A number of physical properties of lysosomes were deduced from the behaviour of acid hydrolases in these experiments. The hepatoma lysosomes seem to have a larger sucrose space than the liver organelles. Injection of Triton WR 1339 , a non-ionic detergent, causes a distinct decrease in the equilibrium density of hepatoma lysosomes in a sucrose gradient similar to that observed for rat-liver lysosomes. 4. 4 . Free activity of acid phosphatase, a reference enzyme for lysosomes, is increased by lowering the tonicity of the medium in which the granules are suspended and also by incubating the particles at pH 5 and 37 °C in isotonic sucrose. HC hepatoma lysosomes appear to be significantly more resistant to this latter treatment than the granules of the other hepatomas.