Franz Dubois

Effect of various flavonoids on lysosomes subjected to an oxidative or an osmotic stress.

When a light mitochondrial fraction (L fraction) of rat liver is incubated in the presence of an oxygen free radical generating system (xanthine-xanthine oxidase), the free activity of N-acetylglucosaminidase (NAGase) increases as a result of the deterioration of the lysosomal membrane. Various flavonoids are able to prevent this phenomenon, others are ineffective. Comparative activity studies suggest the importance of the presence of two OH groups in orthosubstitution in the B ring and of an OH in the 3 position. Flavan-type flavonoids behave like their related flavonoids; d-catechin also opposes lysosome disruption. Kaempferol, quercetin, 7,8-dihydroxyflavone and d-catechin inhibit lipoperoxidation occurring in an L fraction incubated with the xanthine oxidase system as ascertained by malondialdehyde (MDA) production. For kaempferol and quercetin, such an inhibition parallels the prevention of NAGase release; this is not the case for the two other compounds where inhibition of NAGase release takes place at a flavonoid concentration lower than that required to oppose MDA production. Morphological observations performed on purified lysosomes confirm the biochemical results. Some flavonoids are also able to prevent release of NAGase caused by the incubation of an L fraction in isoosmotic glucose. Only flavone and hydroxyflavones are effective. It is proposed that the protective effect of flavonoids on lysosomes subjected to oxygen free radicals does not only originate from their scavenger and antilipoperoxidant properties; a more direct action on lysosomal membrane making it more resistant to oxidative aggression has to be considered. The prevention by some flavonoids of lysosome osmotic disruption in isoosmotic glucose could be the result of an inhibition of glucose translocation through the lysosomal membrane.

Lateral phase separations and structural integrity of the inner membrane of rat-liver mitochondria: Effect of compression. Implications in the centrifugation of these organelles

When maintained in the vicinity of the lower transition temperature of their membrane lipids, rat-liver mitochondria undergo lysis as shown by the release of malate dehydrogenase, (an enzyme located within the mitochondrial matrix), in the surrounding medium. Structural changes take place in the membranes of mitochondria subjected to increasing pressure at 0 degrees C, when the pressure reaches 750 kg/cm2. Freeze-fracture electron microscopy shows the appearance of smooth areas devoid of particles in fracture faces of mitochondrial membranes, together with zones, where aggregated particles can be seen. Concurrently, a suppression of the malate dehydrogenase structure-linked latency is observed. These structural changes can be prevented by increasing the temperature at which compression is performed. The freeze-etching observations suggest that lateral phase separations occur in mitochondrial membranes subjected to high pressure. This can be explained by supposing that pressure promotes the gel-phase appearance in a lipid system and raises the transition temperature since the transition liquid crystal lead to gel is accompanied by a decrease in volume. The deterioration of mitochondria subjected to high pressure is interpreted as a result of the lateral phase separation induced by compression in the membranes. These results are discussed with respect to our interpretation of the damaging effects that hydrostatic pressure, generated by centrifugation, exerts on rat-liver mitochondria.

Subcellular Localization of Mannose 6-Phosphate Glycoproteins in Rat Brain

The intracellular transport of soluble lysosomal enzymes relies on the post-translational modification ofN-linked oligosaccharides to generate mannose 6-phosphate (Man 6-P) residues. In most cell types the Man 6-P signal is rapidly removed after targeting of the precursor proteins from the Golgi to lysosomes via interactions with Man 6-phosphate receptors. However, in brain, the steady state proportion of lysosomal enzymes containing Man 6-P is considerably higher than in other tissues. As a first step toward understanding the mechanism and biological significance of this observation, we analyzed the subcellular localization of the rat brain Man 6-P glycoproteins by combining biochemical and morphological approaches. The brain Man 6-P glycoproteins are predominantly localized in neuronal lysosomes with no evidence for a steady state localization in nonlysosomal or prelysosomal compartments. This contrasts with the clear endosome-like localization of the low steady state proportion of mannose-6-phosphorylated lysosomal enzymes in liver. It therefore seems likely that the observed high percentage of phosphorylated species in brain is a consequence of the accumulation of lysosomal enzymes in a neuronal lysosome that does not fully dephosphorylate the Man 6-P moieties.

Characterization of a Neutral Endopeptidase Localized in the Mitochondrial Matrix of Rat Anterior Pituitary Tissue with GnRH as a Substrate

We have determined the subcellular localization of an endopeptidase activity able to degrade gonadotropin releasing hormone (GnRH) and present in the rat adenohypophysis. After fractionation of tissue homogenates in 0.25 M sucrose by differential centrifugation, about 25% of the total cellular GnRH degrading activity was found to be sedimentable and recovered from heavy (M) and light (L) mitochondrial fractions with a distribution pattern similar to that of the mitochondrial and lysosomal reference enzymes cytochrome oxidase and beta-galactosidase. Upon further fractionation on sucrose density gradients, the activity comigrated with mitochondria. The peptidase appears endowed with a structure-linked latency; the activity is low in a freshly prepared mitochondrial fraction and increases upon treatment with membrane disrupting agents in a manner similar to that of malate dehydrogenase, a component of the mitochondrial matrix. Determination of GnRH cleavage sites was performed by amino acid analysis of the fragments obtained after incubation of the peptidase with (3H)-GnRH labelled on the pyroglutamic acid residue, in presence of carboxypeptidase and peptidyldipeptidase inhibitors. The fragments were separated by ion-exchange chromatography on an Aminex Q-15S column and purified by chromatography on silica gel plates. Fragments 1-2, 1-3, 1-4, 1-5 and 1-6 were all present as early as 1 min after the beginning of incubation. Formation of each of them was inhibited to the same extent by EDTA, mersalyl acid, dithioerythritol and Na deoxycholate. The same fragmentation pattern was observed after partial purification of the enzyme by gel filtration. These data indicate that cleavage of several peptide bonds may result from a possibly single endopeptidase located in the mitochondrial matrix space.

Permeability of mitochondria to sucrose induced by hydrostatic pressure.

When subjected to increasing pressure at 0 degree C, rat liver mitochondria become permeable to sucrose, causing them to swell and their outer membrane to rupture. Afterwards they are lysed and their matrix content is released into the medium. This permeation to sucrose may be prevented to some extent by increasing the temperature at which compression is carried out. 0.75 mM imipramine protects mitochondria against lysis caused by hydrostatic pressure, but does not oppose their permeation to sucrose nor the swelling resulting from the compression. At this concentration, the drug does not exhibit a significant effect on the lateral phase separations which take place in the inner mitochondrial membrane under pressure. The mitochondria of rat fetal liver (21 days), kidney and Morris hepatoma 16 become permeable to sucrose when they are subjected to compression; under these conditions, lateral phase separations occur in their inner membrane. Contrary to liver mitochondria, the former do not undergo lysis. Taking into account both present and previous results, events leading to mitochondrial membrane deterioration by hydrostatic pressure may be summarized in the following way. Pressure first leads to a phase transition of the membrane lipids, thus causing a permeation to sucrose; as a result the mitochondria swell because they have absorbed osmotic water. The membrane lipids freeze increasingly as the pressure increases; the inner membrane becomes fragile and finally, in the case of the adult liver organelles, can no longer resist the swelling. All these events can be avoided by increasing the temperature; imipramine only prevents inner membrane lysis.

Differences in the cellular location of substances endocytosed by rat liver as observed from the distribution patterns obtained after isopycnic centrifugation in a sucrose gradient

We have investigated the distribution of several substances endocytosed by rat-liver, after isopycnic centrifugation in a sucrose gradient of the MLP fractions (de Duve, Pressman, Gianetto, Wattiaux and Appelmans (1955) Biochem.J. 63, 604-617) isolated at increasing times after injection. It has been observed that there are changes in the distribution pattern with time depending on whether the substance is taken up by parenchymal or sinusoidal cells. The results suggest that centrifugation experiments can be informative with respect to the cellular location of a molecule endocytosed by the liver.

Fate of asialofetuin endocytosed by rat liver

We have investigated the endocytosis by rat liver of asialofetuin coupled to [125I] tyramine cellobiose: [125I] TCASF. Subcellular distribution of radioactive compounds was established after differential and isopycnic centrifugation and by analysing the fractions by SDS electrophoresis. Labelling secondary lysosomes was performed by injecting rats with Triton WR 1339 four days before injecting the protein. Results show that after being associated with endosomes [125I] TCASF is recovered in organelles where they are subjected to a first degradation, the density of these organelles is practically not affected by Triton WR 1339 injection. Later the degradation products are associated with lysosomes whose density is markedly lowered by Triton WR 1339 treatment. These observations suggest that the first intracellular organelles where [125I] TCASF is subjected to digestion are distinct from the secondary lysosome population. This could be in agreement with the hypothesis that supposes that endosomes acquire enzymes from primary lysosomes before fusion with secondary lysosomes.

Subcellular distribution of adenylate cyclase in rat-liver tissue

It is generally assumed that adenylate cyclase is located in the plasma membrane. In rat liver however multiple subcellular localizations of this enzyme have been described. According to [l] more adenylate cyclase could be associated with the endoplasmic reticulum than with the plasma membrane, whereas in [2] the highest specific activity of the enzyme was found in Golgi membrane preparations. The possible multiplicity of the subcellular localization of this enzyme should be considered with respect to its function in the cell and its utilization as a specific membrane marker. We have reinvestigated the intracellular localization of adenylate cyclase in rat liver by making use of the analytical approach of centrifugation. Such an approach allows the distribution pattern of an enzyme in different centrifugation systems to be established and when compared with that of the reference enzymes in the same systems, to propose the most plausible subcellular localization of this enzyme. More the centrifugation systems are numerous more the information is precise. Here, the distribution of adenylate cyclase was established after differential and gradient centrifugations of homogenates and particulate fractions. Adenylate cyclase distribution has been compared with the distribution of markers for the plasma membrane (S’nucleotidase), Golgi membranes (galactosyl transferase) and endoplasmic reticulum (glucose 6-phosphatase). We show that when rat-liver adenylate cyclase is stimulated by fluoride, its behaviour in different centrifugation systems is similar to that of an enzyme quasiexclusively associated with the plasma membrane.

Submitochondrial localization of DNA polymerase in rat liver tissue.

The submitochondrial localization of rat liver mitochondrial DNA polymerase is not well established. A report of Schultz and Nass indicates that after treatment of mitochondria with deoxycholate, the specific activity of endogenously primed DNA polymerase is higher in the membrane components than in the whole mitochondria [ 11. The results presented here show that mitochondrial DNA polymerase is fured to the inner side of the granule inner membrane.