Purification and characterization of human lysosomes from EB-virus transformed lymphoblasts

A method was developed for the isolation of unmodified lysosomes of human origin using cultured EB-virus transformed lymphoblasts. The cells were lysed carefully by repeated resuspension in buffered isotonic sucrose. A crude granular fraction derived from this lysate was further purified by isopyknic centrifugation in an isotonic colloidal silica gel gradient and by free-flow electrophoresis. The following relative specific activities (mean ± S.D.) of lysosomal marker enzymes were measured in a pooled lysosomal fraction obtained from the final electrophoresis step (representing less than 0.1% of the initial protein): β-N-acetylglucosaminidase 85.6 ± 15.5; β-galactosidase 87.6 ± 13.4; acid β-glycerophosphatase 41.7 ± 3.5; β-glucuronidase 36.6 ± 6.1. With respect to the final two enzymes the recovery within this pooled fraction was 5–6% of the initial lysate. The great differences in relative specific activities achievable may be due mainly to different extralysosomal portions of the lysosomal marker enzymes, as was found for acid β-glycerophosphatase which was largely distributed within non-lysosomal structures in lymphoblasts when studied by histochemical staining. The final fraction consisted almost exclusively of lysosomes when examined by electron microscopy. Most lysosomes appeared club-shaped immediately after cell lysis and throughout the preparation procedure. Examination by electron microscopy and measurement of the latency of lysosomal enzyme activity revealed an exceptional integrity of the lysosomal membrane. This method provides the opportunity to study highly purified lysosomes from patients with lysosomal disorders.     

Intercellular exchange of lysosomal hydrolases between mutant human fibroblasts and other cell types

Human fibroblasts with a genetic deficiency of a single lysosomal enzyme and fibroblasts from a patient with ‘I-cell’ disease with a multiple deficiency of lysosomal hydrolases were used as recipient cells in studies on recognition and uptake of β-N-acetylhexosaminidase (hexosaminidase), β-glucuronidase and β-galactosidase. Normal human fibroblasts, and fibroblasts, hepatocytes and hepatoma cells from the rat were used as donor cells. The release of hexosaminidase was found to be similar among these different cell types, but the extracellular activities of β-glucuronidase and β-galactosidase were much higher in the rat cell cultures than in cultures of normal human fibroblasts. The enzymes released by rat fibroblasts were ingested by deficient human fibroblasts; enzyme from normal human fibroblasts was shown to be taken up by rat fibroblasts by means of electrophoresis. This indicates that reciprocal transfer of lysosomal hydrolases occurs between human and rat fibroblasts. Rat hepatocytes released hydrolases that were poorly taken up by human recipient fibroblasts and uptake of human fibroblast enzyme was not detected in the hepatocytes. Rat hepatoma cells, on the other hand, released lysosomal enzymes that were taken up by human deficient cells with a higher efficiency than those from fibroblasts. The uptake was subject to competitive inhibition by mannose 6-phosphate, the kinetics of which were comparable with those reported for ‘high-uptake’ forms of lysosomal enzymes [1–2]. Electrophoretic studies showed that rat hepatoma cells were not only capable of ingesting hexosaminidase from normal human fibroblasts, but also defectively processed enzyme [4–5] released by ‘I-cells’. These findings make rat hepatoma cells a useful model for the study of recognition and uptake of lysosomal enzymes.     

A new method for the preparation of rat liver lysosomes. Separation of cell organelles of rat liver by carrier-free continuous electrophoresis

A combination of differential centrifugation and carrier-free continuous electrophoresis is introduced as a new method for the isolation of animal cell organelles. Various buffers were systematically checked in order to find the system which preserves the organelles and gives as well a good separation in the free-flow electrophoresis apparatus. Triethanolamine-acetate buffer (10 mM), pH 7.4 was used. The isolated lysosomes were pure according to marker enzymes and electron micrographs. A heterogeneity of the lysosomes in electrophoretic mobility was demonstrated with respect to the marker enzymes arylsulfatase and β-glucuronidase. The lysosomes with higher mobility showed a maximum enrichment of 240-fold with respect to arylsulfatase. The lysosomes with lower electrophoretic mobility showed a 65-fold enrichment with respect to β-glucuronidase. The ratio of β-glucuronidase to arylsulfatase varied from 2:1 to 1:2 in lysosomes of different mobility. The yield amounted to approximately 1 mg of lysosomal protein per gram of liver protein. 5–8 mg of lysosomes can be obtained in one experiment. The electrophoretic separation proves to be an effective tool in obtaining pure and well preserved lysosomes.     

Rat-kidney lysosomes: isolation and properties

1. The activities of lysosomal enzymes in the cortexes and medullas and the principal subcellular fractions of rat kidney were measured. 2. A method is described for the isolation of rat-kidney lysosomes and a detailed analysis of the enzymic composition of the lysosomes is reported. Enzyme analysis of the other principal subcellular fractions is included for comparison. 3. Studies of the distribution of α-glucosidase showed that the lysosomal fraction contained only 10% of the total enzyme activity. The microsomal fraction contained most of the particulate α-glucosidase. Lysozyme was concentrated mainly in the lysosomal fraction with only small amounts present in the microsomal fraction. Lysosomal α-glucosidase had optimum pH5 whereas the microsomal form had optimum pH6. Both lysosomal and microsomal lysozyme had optimum pH6·2. 4. The stability of lysosomal suspensions was studied. Incubation at 37° and pH7 resulted in first an increased availability of enzymes without parallel release of enzyme. This was followed by a second stage during which the availability of enzymes was closely related to the release of enzymes. These changes were closely paralleled by changes in light-scattering properties of lysosomes. 5. The latent nature of the α-glucosidase and lysozyme of intact kidney lysosomes was demonstrated by their graded and parallel release with other typical lysosomal enzymes. 6. Isolated lysosomes were unstable at pH values lower than 5, most stable at pH6–7 and less stable at pH 8–9. Lysosomes were not disrupted when the osmolarity of the suspending medium was decreased from 0·6m to 0·25m. 7. The discussion compares the properties and composition of kidney lysosomes, liver lysosomes and the granules of macrophages. 8. The possible origin of the lysozyme in kidney lysosomes by reabsorption of the lysozyme in blood is discussed.     

Cell surface-associated lysosomal enzymes in cultured human skin fibroblasts

Trypsin released from the surface of intact human skin fibroblasts β-N-acetylglucosaminidase. The amount of trypsin removable β-N-acetylglucosaminidase in 4 control and 14 mucopolysaccharidosis cell lines was equivalent to 1.5% (range 0.5–4.3%) of the intracellular activity. Cell surface-associated β-N-acetylglucosaminidase was absent in mucolipidosis II and III fibroblasts that form lysosomal enzymes defective in binding to the cell surface receptors of fibroblasts and in β-N-acetylglucosaminidase deficient fibroblasts (Sandhoff's disease). Indirect immunofiuorescence with monospecific antisera allowed the demonstration of β-N-acetylglucosaminidase, α-N-acetylglucosaminidase, α-mannosidase and β-glucuronidase on the cell surface of fibroblasts, whereas these enzymes were absent on the cell surface of mucolipidosis II and III fibroblasts. Simultaneous staining for β-glucuronidase and β-N-acetylglucosaminidase showed presence of both enzymes in almost identical areas of the same cell. Cross-reacting material was present on the cell surface of fibroblasts with a deficiency of β-N-acetylglycosaminidase, α-N-acetylglucosaminidase (mucopolysaccharidosis III B), α-mannosidase (mannosidosis) and β-glucuronidase (mucopolysaccharidosis VII). The demonstration of lysosomal enzymes on the cell surface is in agreement with the hypothesis that in fibroblasts transport of lysosomal enzymes to the lysosomal apparatus involves cycling of lysosomal enzymes via the cell surface.     

Biological Time-Related Changes in Tolerance of Male Mice to Hypoxia—II. Orcadian Rhythm of Lysosomal Susceptibility to Hypoxia

Circadian variations of mouse liver, brain and heart lysosomal susceptibility to hypoxia were investigated. Lysosomal disruption during hypoxia was estimated on the basis of the following measurements: changes in percentage free activity of β-galactosidase and acid phosphatase, tissue loss of both lysosomal enzymes and accumulation of serum β-galactosidase. When exposure to hypoxia took place at the end of the rest phase or at the beginning of the active phase, it was accompanied by maximum increase of percent free activity. This, presumably represents a diffusion of enzymes from lysosomes due to altered membrane permeability. However, hypoxia when occurring during the second part of the active phase and first part of the rest phase resulted in tissues loss of lysosomal enzymes and accumulation of serum lysosomal enzymes. This is believed to represent the release of lysosomal enzymes in bulk from damaged or ruptured lysosomal membranes.     

Membrane cholesterol regulates lysosome-plasma membrane fusion events and modulates Trypanosoma cruzi invasion of host cells

Background

Trypomastigotes of Trypanosoma cruzi are able to invade several types of non-phagocytic cells through a lysosomal dependent mechanism. It has been shown that, during invasion, parasites trigger host cell lysosome exocytosis, which initially occurs at the parasite-host contact site. Acid sphingomyelinase released from lysosomes then induces endocytosis and parasite internalization. Lysosomes continue to fuse with the newly formed parasitophorous vacuole until the parasite is completely enclosed by lysosomal membrane, a process indispensable for a stable infection. Previous work has shown that host membrane cholesterol is also important for the T. cruzi invasion process in both professional (macrophages) and non-professional (epithelial) phagocytic cells. However, the mechanism by which cholesterol-enriched microdomains participate in this process has remained unclear.

Methodology/Principal Finding

In the present work we show that cardiomyocytes treated with MβCD, a drug able to sequester cholesterol from cell membranes, leads to a 50% reduction in invasion by T. cruzi trypomastigotes, as well as a decrease in the number of recently internalized parasites co-localizing with lysosomal markers. Cholesterol depletion from host membranes was accompanied by a decrease in the labeling of host membrane lipid rafts, as well as excessive lysosome exocytic events during the earlier stages of treatment. Precocious lysosomal exocytosis in MβCD treated cells led to a change in lysosomal distribution, with a reduction in the number of these organelles at the cell periphery, and probably compromises the intracellular pool of lysosomes necessary for T. cruzi invasion.

Conclusion/Significance

Based on these results, we propose that cholesterol depletion leads to unregulated exocytic events, reducing lysosome availability at the cell cortex and consequently compromise T. cruzi entry into host cells. The results also suggest that two different pools of lysosomes are available in the cell and that cholesterol depletion may modulate the fusion of pre-docked lysosomes at the cell cortex.     

Copper-independent lysosomal localisation of the Wilson disease protein ATP7B

In hepatocytes, the Wilson disease protein ATP7B resides on the trans-Golgi network (TGN) and traffics to peripheral lysosomes to export excess intracellular copper through lysosomal exocytosis. We found that in basal copper or even upon copper chelation, a significant amount of ATP7B persists in the endolysosomal compartment of hepatocytes but not in non-hepatic cells. These ATP7B-harbouring lysosomes lie in close proximity of ~10 nm to the TGN. ATP7B constitutively distributes itself between the sub-domain of the TGN with a lower pH and the TGN-proximal lysosomal compartments. The presence of ATP7B on TGN-lysosome colocalising sites upon Golgi disruption suggested a possible exchange of ATP7B directly between the TGN and its proximal lysosomes. Manipulating lysosomal positioning significantly alters the localisation of ATP7B in the cell. Contrary to previous understanding, we found that upon copper chelation in a copper-replete hepatocyte, ATP7B is not retrieved back to TGN from peripheral lysosomes; rather, ATP7B recycles to these TGN-proximal lysosomes to initiate the next cycle of copper transport. We report a hitherto unknown copper-independent lysosomal localisation of ATP7B and the importance of TGN-proximal lysosomes but not TGN as the terminal acceptor organelle of ATP7B in its retrograde pathway.     

Tumor necrosis factor-alpha-associated lysosomal permeabilization is cathepsin B dependent

Cathepsin B (Cat B) is released from lysososomes during tumor necrosis factor-alpha (TNF-alpha) cytotoxic signaling in hepatocytes and contributes to cell death. Sphingosine has recently been implicated in lysosomal permeabilization and is increased in the liver by TNF-alpha. Thus the aims of this study were to examine the mechanisms involved in TNF-alpha-associated lysosomal permeabilization, especially the role of sphingosine. Confocal microscopy demonstrated Cat B-green fluorescent protein and LysoTracker Red were both released from lysosomes after treatment of McNtcp.24 cells with TNF-alpha/actinomycin D, a finding compatible with lysosomal destabilization. In contrast, endosomes labeled with Texas Red dextran remained intact, suggesting lysosomes were specifically targeted for permeabilization. LysoTracker Red was released from lysosomes in hepatocytes treated with TNF-alpha or sphingosine in Cat B(+/+) but not Cat B(-/-) hepatocytes, as assessed by a fluorescence-based assay. With the use of a calcein release assay in isolated lysosomes, sphingosine permeabilized liver lysosomes isolated from Cat B(+/+) but not Cat B(-/-) liver. C(6) ceramide did not permeabilize lysosomes. In conclusion, these data implicate a sphingosine-Cat B interaction inducing lysosomal destabilization during TNF-alpha cytotoxic signaling.     

Role for LAMP‐2 in endosomal cholesterol transport

The mechanisms of endosomal and lysosomal cholesterol traffic are still poorly understood. We showed previously that unesterified cholesterol accumulates in the late endosomes and lysosomes of fibroblasts deficient in both lysosome associated membrane protein-2 (LAMP-2) and LAMP-1, two abundant membrane proteins of late endosomes and lysosomes. In this study we show that in cells deficient in both LAMP-1 and LAMP-2 (LAMP^−/−), low-density lipoprotein (LDL) receptor levels and LDL uptake are increased as compared to wild-type cells. However, there is a defect in esterification of both endogenous and LDL cholesterol. These results suggest that LAMP^−/− cells have a defect in cholesterol transport to the site of esterification in the endoplasmic reticulum, likely due to defective export of cholesterol out of late endosomes or lysosomes. We also show that cholesterol accumulates in LAMP-2 deficient liver and that overexpression of LAMP-2 retards the lysosomal cholesterol accumulation induced by U18666A. These results point to a critical role for LAMP-2 in endosomal/lysosomal cholesterol export. Moreover, the late endosomal/lysosomal cholesterol accumulation in LAMP^−/− cells was diminished by overexpression of any of the three isoforms of LAMP-2, but not by LAMP-1. The LAMP-2 luminal domain, the membrane-proximal half in particular, was necessary and sufficient for the rescue effect. Taken together, our results suggest that LAMP-2, its luminal domain in particular, plays a critical role in endosomal cholesterol transport and that this is distinct from the chaperone-mediated autophagy function of LAMP-2.     

Intracellular transport and degradation of I-labelled denatured serum albumin in isolated nonparenchymal rat liver cells

The intracellular movement, following uptake of ¹²⁵I-labelled denatured serum albumin into nonparenchymal liver cells, was followed by means of subcellular fractionation. Isolated nonparenchymal rat liver cells were prepared by means of differential centrifugation. The cells were homogenized in a sonifier and the cytoplasmic extract subjected to isopycnic centrifugation in a sucrose gradient. The intracellular movement of the labelled albumin was followed by comparing the distribution profile of radioactivity in the sucrose gradient with those of marker enzymes for plasma membrane and lysosomes. The distribution profiles for radioactivity after the cells had been exposed to the labelled denatured albumin for different time periods indicated that the radioactivity was first associated with subcellular fractions of lower modal densities than the lysosomes. With time of incubation the radioactivity moved towards higher densities. After prolonged incubations in the absence of extracellular labelled denatured albumin the radioactivity peak coincided with that of the lysosomal marker β-acetylglucosaminidase. When the cells were treated with the lysosomal inhibitor leupeptin, degradation of the labelled albumin was decreased, resulting in a massive intracellular accumulation of radioactivity. The radioactivity peak coincided with the peak of activity for the lysosomal marker β-acetylglucosaminidase, suggesting lysosomal degradation.     

Isolation of autofluorescent ‘aged’ human fibroblasts by flow sorting : Morphology, enzyme activity and proliferative capacity

In cultures of human fibroblasts the percentage of bright autofluorescent (AF) cells increases with increasing passage number. These autofluorescent cells were isolated using a FACS II cell sorter and compared with sorted non-fluorescent (NF) cells. The AF cells showed an increase in population doubling time (2.3-fold), cell protein (1.9-fold), and in specific activities of the lysosomal enzymes: β-hexosaminidase (4.2-fold), β-galactosidase (3.8-fold) and acid phosphatase (2.5-fold). The specific activities of two non-lysosomal enzymes glucose-6-phosphate dehydrogenase and lactate dehydrogenase had increased only slightly (1.1-fold) respectively (1.5-fold).The autofluorescence in the AF cells was restricted to small round organelles. The distribution and size of these autofluorescence granules were similar to the acid phosphatase-containing granules in the cytochemically stained cells. Electronmicroscopical examination showed that these AF cells contained a large amount of small electron-dense granules containing amorphosmophilic material. These granules which were positive for the acid phosphatase reaction, were classified as secondary lysosomes. The low percentage of the sorted AF cells which incorporate [³H]thymidine during a 24 h test period (19%) as compared with the labelling percentage of sorted NF cells (73%) from the same culture, indicate that the autofluorescent cells in a ‘young’ culture have a very limited remaining proliferative capacity. The results imply, that by flow sorting it is possible to isolate ‘aged’ cells with characteristics of ‘phase III’ cells out of non-aged fibroblast cultures.     

Properties of the lysosomes from liver and gill of rainbow trout,Salmo gairdnerii R.: Effect of starvation,salinity and 2, 4, 5-T

1. Three isolation procedures were used to test the labilization of the lysosomes after adaptation of the animals to seawater, starvation or acute and chronic treatments with 2, 4, 5-T, an organochlorine pesticide.2. The lysosomes from gill and liver had different properties with respect to their resistance to osmotic and mechanical shocks, or treatments with digitonin.3. Starvation induced a significant labilization of the lysosomes in liver, but not in gill.4. Salinity changes were without effect on the stability of the gill lysosomes, but induced an increase of the specific activity of the lysosomal enzyme β-acetylglucosaminidase.5. Acute treatments with 2, 4, 5-T increased lysosomal fragility in the gill but not in the liver, while chronic treatments with this herbicide increased lysosomal lability in the two tissues.6. Liver lysosomes were much more susceptible toin vitro treatments with 2, 4, 5-T than gill lysosomes.7. The results are discussed with respect to the different functions of gill and liver, their exposure to the environment, and the possible discrimination between different lysosomal populations.     

Changes in the composition of the lysosomes in hepatocytes cultured in vitro in modelling pathological states

The monolayer cultures of newborn rats hepatocytes were treated with substrate starvation (20 min.) anoxia accompanied with substrate starvation (1 h.), following rehabilitation (1 h.) and cooling (4 degrees C, 30 min). After the neutral red staining we have examined the alterations in lysosomes quantity and the development of phagocytosis; we also tested the hepatocytes distribution due to the amount of the second lysosomes per cell. We have shown that a short time pathological treatment caused a decrease in large lysosomes (1-3 microns) amount in significant portion of hepatocytes. There were neither increase in phagosomes amount per cell nor alterations in the quantity of cells, containing phagosomes. However a long time pathological treatment caused the increase in phagosomes and cells. We propose that initial stage of lysosomal apparatus alterations, connected with reduction of CAMP level, is accompanied by a stamping of large lysosomes.     

An electron microscope autoradiographic study of the carbohydrate recognition systems in rat liver. II. Intracellular fates of the 125I- ligands

Electron microscope autoradiographic and biochemical methods were used to study the intracellular fates of several 125I-glycoproteins, known to be specifically bound and internalized by the different cell types in the liver. At the earliest times examined (1--2 min), 125I-glycoproteins (ASGP) were localized predominantly along the sinusoidal front of hepatocytes. Analysis of the distribution of autoradiographic grains indicated that: (a) approximately 40--60% of the 125I-ligand could be ascribed to the plasmalemma; (b) a significant fraction had already been internalized; yet (c) very little 125I-ligand was present in the lysosome-Golgi region. Between 4 and 15 min after administration of 125I-ASGPs, there was a dramatic redistribution of autoradiographic grains from regions of the plasmalemma and peripheral cytoplasm (30% decrease) to the lysosome-Golgi region (30% increase). At longer times (30 min), there was continued drainage of 125I-ASGP into this region. The grain density over secondary lysosomes was 60--90 times higher than that over recognizable Golgi elements, clearly indicating that lysosomes were the ultimate destination of the 125I-ASGP. However, no more than 60% of the total 125I-ligand could be localized to lysosome-rich regions of the hepatocyte, with the remaining 40% primarily in the intermediate cytoplasm. Biochemical evidence for proteolysis of the internalized 125I-ASGP (presumably within lysosomes) was obtained when [125I]-mono-iodotyrosine was found in the liver (i.e., hepatocytes) at times later than 15 min. The temporal redistribution observed for mannose and N-acetylglucosamine-terminated glycoproteins (ahexosamino-orosomucoid and agalacto-orosomucoid, respectively) in endothelial cells indicated that the 125I-ligands resided in macropinocytic vesicles (1--15 min) before their ultimate residence in dense bodies (15 min). The same 125I-ligands were also localized to structures resembling secondary lysosomes in Kupffer cells. The lysosomal nature of "these organelles" was implied from the appearance of [125I]mono-iodotyrosine in the liver at later times. 125I-beta-glucuronidase followed the same intracellular pathway in both cell types but was not degraded.     

Fate of N-(2-hydroxypropyl)methacrylamide copolymers with pendent galactosamine residues after intravenous administration to rats

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers bearing galactosamine residues accumulate in the liver after intravenous administration to rats (Duncan, R., Kopec̆ek, J., Rejmanová, P. and Lloyd, J.B. (1983) Biochim. Biophys. Acta 755, 518–521). In this study HPMA copolymers bearing pendent galactosamine residues (1.0–11.6 mol%) were injected intravenously intor rats and their rates of blood clearance and liver accumulation were measured. A level of substitution of 4 mol% was found to be sufficient to cause substantial deposition in the liver 30 min after administration. The most highly substituted polymer (11.6 mol%) was directed rapidly to the liver, 80–90% being recovered there less than 10 min after administration. Separation of liver into hepatocytes and non-parenchymal cells indicated that polymer was largely associated with the hepatocytes, and density-gradient subcellular fractionation of liver at various times after administration confirmed that polymer was internalized by liver cells and transported, with time, into the secondary lysosomes. Experiments using isolated rat hepatocytes indicated that HPMA copolymers with high galactosamine content have higher affinity for the hepatocyte plasma membrane. HPMA copolymers containing galactosamine and in addition glycylglycyltyrosianmide side-chains were used to demonstrate release of a drug analogue across the lysosomal membrane. These polymers were radioiodinated and, following intravenous administration to rats, the liver lysosomes were isolated and incubated at 37°C in 0.25 M sucrose. Radioactivity was released from the lysosomes faster than the lysosomal enzyme arylsulphatase, an observation that indicates intralysosomal hydrolysis of the copolymer side-chain with subsequent passage of low molecular weight degradation product across the lysosomal membrane.     

Characterisation of lipofuscin-like lysosomal inclusion bodies from human placenta

A structural hallmark of lysosomes is heterogeneity of their contents. We describe a method for isolation of particulate materials from human placental lysosomes. After a methionine methyl ester-induced disruption of lysosomes and two density gradient centrifugations we obtained a homogeneous membrane fraction and another one enriched in particulate inclusions. The latter exhibited a yellow-brown coloration and contained bodies lacking a delimiting membrane, which were characterised by a granular pattern and high electron density. The lipofuscin-like inclusion materials were rich in tripeptidyl peptidase I, beta-glucuronidase, acid ceramidase and apolipoprotein D and contained proteins originating from diverse subcellular localisations. Here we show that human term placenta contains lipofuscin-like lysosomal inclusions, a phenomenon usually associated with senescence in postmitotic cells. These findings imply that a simple pelleting of a lysosomal lysate is not appropriate for the isolation of lysosomal membranes, as the inclusions tend to be sedimented with the membranes.     

Thyroid lysosomes: the stability of the lysosomal membrane

To determine the integrity of lysosomes during their isolation from rat thyroid glands and their subsequent incubation at 37 degrees C, the sedimentability of lysosomal acid phosphatase and thyroglobulin (amount of undisrupted lysosomes) and the latency of sedimentable acid phosphatase (permeability of undisrupted lysosomes) were measured concomitantly. The following results were obtained: (a) During isolation of lysosomes in 0.25 M sucrose medium, mild homogenization of thyroid tissue or cholesterol addition did not modify the amount of undisrupted lysosomes but reduced their permeability. Homogenization in 0.5 M sucrose decreased both the amount and the permeability of undisrupted lysosomes. It also reduced their content of recently iodinated thyroglobulin (Tg). Cholesterol addition had no effect in 0.5 M sucrose medium. (b) During incubations at 37 degrees C of lysosomes, the amount of undisrupted lysosomes decreased progressively while their permeability increased. According to the incubation pH, the permeability of lysosomes prepared in 0.25 M sucrose was either more (pH 8) or less (pH 6) extensively increased than that of lysosomes prepared in 0.5 M sucrose. From these results, we concluded: (a) that isolation and incubation of the thyroid lysosomal fraction induce increased permeability of lysosomes prior to their complete disruption: (b) that recently formed lysosomes (high content of recently iodinated Tg) and aged lysosomes (low content of recently iodinated Tg) differ significantly. Recently formed lysosomes are more permeable, are stabilized by cholesterol and are more extensively disrupted in 0.5 M sucrose medium. During incubations, the permeabilities of these two classes of lysosomes are also differently affected by external pH.     

Distribution of organelles and membranes between hepatocytes and nonhepatocytes in the rat liver parenchyma. A stereological study

When biochemical studies on the liver are interpreted, the cells of the sinusoidal area frequently receive little attention because, compared to hepatocytes, their contribution to subcellular fractions is assumed insignificant. A systematic stereological analysis of liver parenchyma was therefore performed in order to determine the distribution of organelles and membranes between hepatocytic and nonhepatocytic cells, namely endothelial, Kupffer, and fat-storing cells. The livers were fixed by vascular perfusion and the data were corrected for systematic errors dur to section thickness and compression. The extracellular space compartment includes the lumina of sinusoids (10.6%), the space of Disse (4.9%), and the bile canaliculi (0.4%). Hepatocytes constitute 78% of parenchymal volume; the nonhepatocytes account for 6.3% and consist of 2.8% endothelial cells, 2.1% Kupffer cells, and 1.4% fat- storing cells. The nonhepatocytes contribute 55% of the volume of lipid droplets in the liver, 43% of the lysosomes, and 1.2% of the mitochondria. Although the nonhepatocytes account for only 8% of the total surface area of parenchymal membranes, they contain 26.5% of all the plasma membranes, 32.4% of the lysosomal membranes, 15.1% of the Golgi apparatus 6.4% of the endoplasmic reticulum, and 2.4% of the mitochondrial membranes. The data demonstrate the extent to which nonhepatocytic organelles can potentially contaminate subcellular fractions used for biochemical studies. Particularly important for the interpretation of studies on lysosomes, plasma membrane, and Golgi apparatus is the finding that an appreciable part of these organelles may be derived from cell types other than hepatocytes.