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At least 19 recordsLinked to original sources

PubMed1990

A cysteine-specific lysosomal transport system provides a major route for the delivery of thiol to human fibroblast lysosomes: possible role in supporting lysosomal proteolysis.

Lysosomes constitute only 4% of the intracellular volume of a normal human fibroblast. When human fibroblasts are incubated for 2-5 min with 20 microM [35S]cystine in Krebs-Ringer phosphate solution at pH 7.4, a minimum of 50-60% of the total radioactivity taken up by the cells is found sequestered into the lysosomal compartment in the form of cysteine. A lysosomal transport system, highly specific for cysteine, appears to facilitate this rapid lysosomal cysteine sequestration. Time courses of [35S]cysteine uptake into isolated, Percoll-purified fibroblast lysosomes at pH 7.0 and 37 degrees C are linear for the first 4-5 min and attain a steady state by 10 min. Lysosomal cysteine uptake displays a Km of 0.05 mM at pH 7.0 and an activation energy of 21 kcal/mol, corresponding to a Q10 of 3.2. The role of this transport system in delivering cysteine into lysosomes is supported by its pH curve showing a slow rate of cysteine transport at the acidic pHs between 5 and 6, but then increasing sevenfold between pH 6 and 7.5 to be maximally active near the cytosolic pH of 7. Carrier mediation by this lysosomal transport route demonstrates a high specificity for cysteine as indicated by the inability of the following amino acids to significantly inhibit at 5 mM the lysosomal uptake of 0.035 mM [35S]L-cysteine: ala, ser, pro, val, gly, homocysteine, D- or L-penicillamine, arg, asp, or leu. Similarly, D-cysteine and beta-mercaptopropionate were poor inhibitors, suggesting that both the L-isomer and alpha-amino group of cysteine appear to be required for recognition by the cysteine-specific transport system. In contrast, cysteamine, which lacks an alpha-carboxyl group, was able to strongly inhibit lysosomal cysteine uptake. The physiological importance of this cysteine-specific lysosomal transport system may be to aid lysosomal proteolysis by delivering cysteine into the lysosomal compartment to (a) maintain the catalytic activity of the thiol-dependent lysosomal enzymes and (b) break protein disulfide bridges at susceptible linkages, thereby allowing proteins to unfold, facilitating their degradation.

Biological Transport↗

PubMed1978

An alternative hypothesis of cellular transport of lysosomal enzymes in fibroblasts. Effect of inhibitors of lysosomal enzyme endocytosis on intra- and extra-cellular lysosomal enzyme activities.

Recapture of lysosomal enzymes secreted by fibroblasts was inhibited by growing the cells in the presence of either free or immobilized antibodies against lysosomal enzymes or in the presence of phosphorylated carbohydrates known to interact with the cell-surface receptors for lysosomal enzymes. The following results were obtained. 1. Conditions that prevent recapture of released lysosomal enzymes increase the rate of extracellular accumulation of these enzymes up to twice that of controls. 2. Growing cells for 12 days in the presence of 0.5mm-mannose 6-phosphate, which decreases beta-N-acetylglucosaminidase endocytosis to less than 10% of that of controls, has no effect on the intracellular activity of this and four other lysosomal enzymes. 3. Growing cells for 4 days in the presence of 50mm-mannose 6-phosphate, which is a 1000-fold higher concentration than that required for 50% inhibition of lysosomal enzyme endocytosis, leads to a 4-fold increase in extracellular beta-N-acetylglucosaminidase accumulation and a decrease in intracellular enzyme. These results give evidence that, in fibroblasts, transfer of lysosomal enzymes into lysosomes does not require secretion before a receptor-mediated recapture [Hickman & Neufeld (1972) Biochem. Biophys. Res. Commun.49, 992-999]. We propose that (a) lysosomal enzymes are present in a receptor-bound form in those vesicles that fuse with the cell membrane, (b) the major part of the lysosomal enzyme cycles via the cell surface in a receptor-bound form and (c) only a minor part of the lysosomal enzyme is released into the extracellular space during its life cycle.

Acetylglucosaminidase↗

PubMed1990

Targeting of a lysosomal membrane protein: a tyrosine-containing endocytosis signal in the cytoplasmic tail of lysosomal acid phosphatase is necessary and sufficient for targeting to lysosomes.

Lysosomal acid phosphatase (LAP) is synthesized as a transmembrane protein with a short carboxy-terminal cytoplasmic tail of 19 amino acids, and processed to a soluble protein after transport to lysosomes. Deletion of the membrane spanning domain and the cytoplasmic tail converts LAP to a secretory protein, while deletion of the cytoplasmic tail as well as substitution of tyrosine 413 within the cytoplasmic tail against phenylalanine causes accumulation at the cell surface. A chimeric polypeptide, in which the cytoplasmic tail of LAP was fused to the ectoplasmic and transmembrane domain of hemagglutinin is rapidly internalized and tyrosine 413 of the LAP tail is essential for internalization of the fusion protein. A chimeric polypeptide, in which the membrane spanning domain and cytoplasmic tail of LAP are fused to the ectoplasmic domain of the Mr 46 kd mannose 6-phosphate receptor, is rapidly transported to lysosomes, whereas wild type receptor is not transported to lysosomes. We conclude that a tyrosine containing endocytosis signal in the cytoplasmic tail of LAP is necessary and sufficient for targeting to lysosomes.

Acid Phosphatase↗

PubMed1983

Degradation of phagocytosed lysosomes by Kupffer cell lysosomes.

Lysosomal membranes are apparently resistant to hydrolytic attack from their own enzymes. Alternatively, degradation occurs but is compensated for by continuous insertion of new membrane components. It may be hypothesized that a mechanism operating exclusively on the luminal side of the lysosomal membrane serves to protect the membrane from being degraded. To evaluate this notion the cytoplasmic side of the lysosomal membrane has been exposed to lysosomal enzymes in vivo. Lysosomes were isolated and subsequently injected into the portal vein of a series of rats. The uptake of the injected organelles by Kupffer cells and their subsequent degradation in lysosomes were monitored by means of electron microscopy. Four minutes after injection lysosomes were seen attached to the surface of the Kupffer cells. After 30 minutes the injected material was present in Kupffer cell phagolysosomes, and signs of degradation of the phagocytosed lysosomes were seen. By 2 hours only a few distinct membranes were left, and by 12 hours the injected lysosomes were no longer recognizable. Instead, the phagolysosomes of Kupffer cells were laden with lipid-like droplets and irregular membranous structures. Acid phosphatase histochemistry and labeling of preexisting Kupffer cell lysosomes with marker particles indicated that the phagosomes engulfing the injected lysosomes acquired hydrolytic enzymes within 30 minutes after their formation. The degradation rate of injected lysosomes was estimated by measuring the decay of radioactivity from a rat liver mitochondrial lysosomal fraction after administration of lysosomes isotopically prelabeled with 14C-leucine and 14C-glycerol. The half-life of the lysosomal membrane proteins varied between 1.5 and 2.0 hours, whereas that of the lipid component was in the range of 2.0 to 3.5 hours. These findings demonstrate that lysosomal membranes are degraded if their outer surface is exposed to lysosomal enzymes. Both the ultrastructural analysis and the isotopic studies indicate that proteins are degraded faster than lipids. Apparently, the cytoplasmic surface of the lysosomes is susceptible to lysosomal hydrolytic attack.

Acid Phosphatase↗

PubMed1983

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.

Acid Phosphatase↗

PubMed1995

Macrophage uptake of oxidized LDL inhibits lysosomal sphingomyelinase, thus causing the accumulation of unesterified cholesterol-sphingomyelin-rich particles in the lysosomes. A possible role for 7-Ketocholesterol.

Macrophage uptake of oxidatively modified LDL (Ox-LDL), unlike the uptake of acetylated LDL (Ac-LDL), resulted in lysosomal accumulation of unesterified cholesterol (UC). As sphingomyelin (SM) binds UC with high affinity, we considered whether lysosomes also accumulate Ox-LDL-derived SM, and if such a phenomenon could be involved in the lysosomal trapping of Ox-LDL-derived UC. Incubation of J-774 A.1 macrophages with Ox-LDL increased the lysosomal accumulations of UC by 75% and SM by 63% compared with the effect of Ac-LDL. The addition of chlorpromazine, an inhibitor of lysosomal sphingomyelinase (SMase), to macrophages that were incubated with [3H]cholesteryl ester-labeled Ac-LDL also led to lysosomal accumulation of both SM and UC. 7-Ketocholesterol (7-KC), the major oxysterol in Ox-LDL, inhibited lysosomal SMase in a cell-free system. The addition of 7-KC to cells in the presence of [3H]choline- or [3H]cholesteryl ester-labeled Ac-LDL led to macrophage accumulation of SM or UC, respectively. Niemann-Pick type C disease (NP-C) is an inherited cholesterol-storage disease in which lysosomal SMase activity is attenuated after uptake of LDL. Incubation of monocyte-derived macrophages from two NP-C patients with Ac-LDL or Ox-LDL resulted in an accumulation of UC in the lysosomes, whereas normal monocyte-derived macrophages accumulate UC in their lysosomes after incubation with Ox-LDL but not Ac-LDL. These results suggest that inhibition of lysosomal SMase in NP-C cells or by 7-KC is required for lysosomal accumulation of UC. Analysis of the macrophage lysosomal extract (following cell incubation with Ox-LDL) by density-gradient ultracentrifugation and gel-filtration chromatography revealed the presence of a particle consisting of UC, SM, 7-KC, and apoB-100. We conclude that 7-KC in Ox-LDL can inhibit lysosomal SMase, thus leading to the accumulation of SM, which binds UC avidly and inhibits its further cellular processing out of the lysosome. As UC-SM particles of lysosomal origin exist in the atherosclerotic lesion, the formation of such particles may result from an impaired processing of Ox-LDL by arterial wall macrophages during early atherogenesis.

Animals↗

PubMed2002

The protein tyrosine kinase Hck is located on lysosomal vesicles that are physically and functionally distinct from CD63-positive lysosomes in human macrophages.

In macrophages, lysosomes are suspected to have a heterogenous population of vesicles. This study was thus undertaken to identify and to characterize lysosomal compartments in human macrophages. Hck is a Src-family tyrosine kinase associated with secretory lysosomes in neutrophils and with cytoplasmic vesicles in macrophages that fuse with phagosomes. We identified these Hck-positive vesicles and compared them to CD63-positive, M6PR-negative vesicles known as classical lysosomes. Hck vesicles exhibited lysosomal features. Indeed, Hck-positive vesicles could be loaded with rhodamine-dextran, which has been shown to accumulate in lysosomal compartments. Hck was delivered to zymosan-containing phagosomes at a late stage of the maturation process, which occurs after the fusion with CD63-positive lysosomes. Finally, when mycobacteria were used to prevent phagolysosome biogenesis, Hck was not recruited to phagosomes. Moreover, Hck lysosomes were physically and functionally distinct from CD63-lysosomes. For instance, sucrose induced swelling of CD63-lysosomes without affecting Hck-positive ones. Only CD63-lysosomes fused with phagosomes in a microtubule-dependent manner. Entry of particles through the mannose receptor and Fcgamma receptors drove the phagosome towards a fusion with CD63-lysosomes, whereas only Fcgamma receptors induced the mobilisation of Hck-lysosomes. This study provides further evidence for the existence of sub-populations of lysosomes in macrophages: one stained by CD63 and another one characterized by the presence of Hck. Therefore, Hck represents a new tool to study the fusion dynamics of lysosomal compartments and their subversion by several intracellular pathogens.

Antigens, CD↗

PubMed1997

Lysosomal biogenesis in lysosomal storage disorders.

Lysosomal biogenesis is an orchestration of the structural and functional elements of the lysosome to form an integrated organelle and involves the synthesis, targeting, functional residence, and turnover of the proteins that comprise the lysosome. We have investigated lysosomal biogenesis during the formation and dissipation of storage vacuoles in two model systems. One involves the formation of sucrosomes in normal skin fibroblasts and the other utilizes storage disorder-affected skin fibroblasts; both of these systems result in an increase in the size and the number of lysosomal vacuoles. Lysosomal proteins, beta-hexosaminidase, alpha-mannosidase, N-acetylgalactosamine-4-sulfatase, acid phosphatase, and the lysosome-associated membrane protein, LAMP-1, were shown to be elevated between 2- and 28-fold above normal during lysosomal storage. Levels of mRNA for the lysosome-associated membrane proteins LAMP-1 and LAMP-2, N-acetylgalactosamine-4-sulfatase, and the 46- and 300-kDa mannose-6-phosphate receptors were also elevated 2- to 8-fold. The up-regulation of protein and mRNA lagged 2-4 days behind the formation of lysosomal storage vacuoles. Correction of storage, in both systems, resulted in the rapid decline of the mRNA to basal levels, with a slower decrease in the levels of lysosomal proteins. Lysosomal biogenesis in storage disorders is shown to be a regulated process which is partially controlled at, or prior to, the level of mRNA. Although lysosomal proteins were differentially regulated, the coordination of these events in lysosomal biogenesis would suggest that a common mechanism(s) may be in operation.

Antibodies, Monoclonal↗

PubMed2002

Isolation of intact lysosomes from human RPE cells and effects of A2-E on the integrity of the lysosomal and other cellular membranes.

BACKGROUND: Accumulation of lipofuscin in RPE cells occurs with age and in association with various retinal diseases. Lipofuscin and its major retinoid compound and fluorophore A2-E interfere with the cellular metabolism of RPE cells in various ways. One of these mechanisms is thought to be related to detergent properties of A2-E. METHODS: We isolated pure and intact lysosomes from RPE cell cultures and investigated detergent-like effects of the lipofuscin compound A2-E on the integrity of lysosomal membrane and other cellular membranes, using latency measurements. A postnuclear supernatant prepared from cultured human RPE cells was used to isolate intact lysosomes by fractionation of cellular organelles in two sequential gradients. Destabilization of the lysosomal membrane was tested by incubating the purified lysosomal fraction in the presence of A2-E and subsequent measurement of the latency of the lysosomal luminal marker beta-hexosaminidase. In order to compare the effect of A2-E on other cellular membranes, latencies of the specific markers succinate dehydrogenase and UDP-galactosyltransferase were assessed using partially purified mitochondria and microsomes. Intactness of the plasma membrane was tested by including A2-E in the culture medium before leakage of lactate dehydrogenase into the medium was determined. RESULTS: A more than 100-fold purification of the lysosomal fraction was achieved. Except for a minor activity of the mitochondrial marker, no contamination with other cell fractions was observed. Intactness of the purified lysosomes was well preserved upon incubations in isotonic media providing the base for investigations on a possible detergent-like action of A2-E on lysosomal integrity. At concentrations above 2 microM A2-E, progressive leakage of the lysosomal marker was observed. In comparison, leakage of the mitochondrial marker was induced at significantly lower concentrations (1 microM), whereas ER/Golgi membranes and the plasma membrane were relatively insensitive to a detergent effect of the retinoid. The described methodology to obtain highly purified and intact lysosomes from RPE cells provides a suitable tool for investigations on compounds affecting lysosomal structure. A2-E was shown to cause desintegration of the lysosomal membrane at relatively low concentrations, which may implicate an involvement of such mechanism in triggering lipofuscin-induced dysfunction of RPE in vivo. Secondary to disintegration of the lysosomal membrane, damage to mitochondria might be an additional pathogenic mechanism. CONCLUSIONS: Our data provide evidence for surfactant-like properties of A2-E on biomembranes which might be operative in retinal diseases associated with excessive lipofuscin-accumulation, such as age-related macular degeneration.

Cell Membrane↗

PubMed1989

Effect of gentamicin on the lysosomal system of cultured human proximal tubular cells. Endocytotic activity, lysosomal pH and membrane fragility.

Gentamicin treatment results in significant changes in lysosomal morphology and enzyme activity in renal tubular epithelium both in vivo and in vitro. In this study, cultured human proximal tubular cells (PTC) were treated with gentamicin (0, 0.01, 0.1, and 1.0 mg/ml) for 3, 7, 10 and 14 days, and the endocytotic activity, pH, and membrane fragility of the lysosomal system were examined. Fluorescein isothiocyanate-labeled dextran (FITC-dextran) was used to estimate endocytotic activity and intralysosomal pH. The fragility of isolated lysosomes was estimated by the release of N-acetyl-beta-glucosaminidase (NAG, EC3.2.1.30) into the medium. Gentamicin content was measured and correlated with the changes seen in lysosomal function. Gentamicin treatment caused a slight decrease in the rate with which human PTC accumulated FITC-dextran and a slight increase in intralysosomal pH. Treatment of human PTC with NH4Cl, a lysosomotropic compound, significantly increased the lysosomal pH; the NH4Cl-induced increase in the lysosomal pH of gentamicin-treated PTC, however, was not significantly different from control (0 mg gentamicin/ml). Lysosomes isolated from human PTC cultures released NAG upon incubation for 60 min at 37 degrees. There was no significant effect on the fragility of lysosomes isolated from cultures exposed to gentamicin for less than or equal to 7 days. Significantly increased fragility was seen, however, after 10 days of treatment with 1.0 mg gentamicin/ml and especially after a 14-day exposure to 0.01, 0.1, and 1.0 mg gentamicin/ml. Human PTC accumulated 0.47, 2.05 and 10.30 micrograms gentamicin/mg protein with 10 days of exposure to 0.01, 0.1 and 1.0 mg gentamicin/ml medium respectively. Gentamicin treatment associated with increased numbers of morphologically altered lysosomes, i.e. myeloid bodies, did not affect significantly the endocytotic activity and pH of lysosomes in cultured human PTC. Prolonged exposure (14 days) of human PTC to gentamicin, however, did increase the fragility of lysosomes after isolation. The increased numbers of morphologically altered lysosomes with increased fragility were not associated with any significant in vitro cell death. Therefore, it would appear that these lysosomal alterations are not directly responsible for the in vivo nephrotoxicity.

Cells, Cultured↗

PubMed1992

The lysosomal membrane glycoprotein lamp-1 is transported to lysosomes by two alternative pathways.

It has been demonstrated that lysosomal membrane proteins are directed to lysosomes by a tyrosine-containing structural motif in their cytoplasmic tails. It is presently unclear whether lysosomal membrane proteins are directly transported to lysosomes or first taken to the plasma membrane and then directed to the lysosomes via the endocytic pathway. In the present study, the transport pathways taken by one of the members of highly glycosylated lysosomal membrane proteins, lamp-1, were examined in human HL-60 cells. Pulse-chase labeling, combined with cell surface biotinylation and Percoll density gradient fractionation, was used to measure the kinetics of transport to the cell surface and lysosomes. The results show that the majority of lamp-1 is directly transported to lysosomes by a fast pathway (half-time 60 min), which involves sorting at an intracellular site, presumably in the trans-Golgi network. A minor part of lamp-1 is transported out to the cell surface, where it is internalized and eventually delivered to lysosomes. Transport by this pathway requires a long transit time (half-time greater than 2 h). After granulocytic differentiation of HL-60 cells by dimethyl sulfoxide, the synthesis of lamp-1 was increased approximately twofold. In these cells, the sorting in the Golgi apparatus is more effective, leaving only a minute fraction of lamp-1 for the bulk flow to the cell surface. This study establishes that the majority of lamp-1 is directly transported to lysosomes and that, in certain cells, the minority of the molecules is transported to lysosomes via the cell surface.

Antigens, CD↗

PubMed1989

Lysosomal movements during heterophagy and autophagy: with special reference to nematolysosome and wrapping lysosome.

Recent studies on lysosomal movements during heterophagy and autophagy performed in our laboratory for the past several years were reviewed; methods for the investigation of lysosomes and the cytoskeleton in these studies mainly involved electron microscopic cytochemistry. Lysosomal movements during heterophagy were observed in cultured rat alveolar macrophages taking up horseradish peroxidase (HRP) and rat peroxidase-antiperoxidase (PAP) by fluid-phase pinocytosis and adsorptive pinocytosis, respectively. A characteristic lysosomal change which was induced by the pinocytosis was the appearance of long, threadlike lysosomes (nematolysosomes) in the cytoplasm. The effects of actin filament destabilizer and antimicrotubular drug on lysosomal changes revealed that the appearance of nematolysosomes was dependent on the presence of both actin filaments and microtubules. The close morphological relationship between lysosomes and cytoskeletal elements, such as actin filaments and microtubules in the alveolar macrophages, supports the participation of the cytoskeletal system in the regulatory mechanism of lysosomal movements. In the study of the lysosomal wrapping mechanism (LWM), which is one type of lysosomal movement that occurs during autophagy, it was found that the occurrence of LWM was dependent on energy--namely, the supply of ATP--and on the presence of actin filaments. However, deconstruction of microtubules induced or favored the occurrence of LWM. It is conceivable that the LWM is also related to the cytoskeletal system. We conclude that intracellular dynamics of lysosomes during heterophagy and autophagy are largely a consequence of complicated modulation by the cytoskeletal system.

Animals↗

PubMed1991

Comparative behavior of lysosomes and the pre-lysosome compartment (PLC) in in vivo cell fusion experiments.

Interspecies cell fusion was used to compare protein intermixing within the mannose 6-phosphate receptor (MPR)-enriched pre-lysosome compartment (PLC) and within the MPR-negative lysosomal compartment. Both compartments were positive for lysosomal glycoprotein (lgp) membrane markers but were morphologically distinct. In most experiments, rat-mouse cell syncytia were formed by u.v.-inactivated Sindbis virus-mediated fusion. By immunogold electron microscopy of syncytia, extensive intermixing of species-specific lysosomal membrane proteins was observed in both lysosomes and PLC. At 3 h post cell fusion, multiple-label immunogold studies showed that 82% of the lysosome-like structures positive for the rat lysosomal membrane protein LIMP-I were also positive for the mouse lysosomal membrane protein mLAMP-1. By immunofluorescence, LIMP-I and mLAMP-1 co-localized with a t1/2 of 30 min after cell fusion; although the lgp-positive organelle populations had evidently interchanged their proteins, the lysosomal structures remained small, punctate bodies distributed throughout the syncytoplasm as observed in single cells. In contrast, the initially separate units of the PLC congregated with a t1/2 of 1 h to form large, pre-lysosome complexes associated with individual nuclear clusters. At the electron-microscope level, gold markers endocytized by the rat and mouse parent cells in a 1 h uptake followed by a 16-20 h chase co-localized in these extended PLC complexes, as did the membrane markers mLAMP-1 and LIMP-I. The density of labeling for rat MPR in the extended PLCs was markedly decreased, consistent with membrane fusions and dilution of the antigen upon congregation of the PLC compartments from the donor cells. The extended PLC complex behaved as a late endocytic compartment, as shown by co-localization of the MPR and rhodamine-dextran following a 10 min dextran uptake and a 50 min chase. These differences in behavior between lysosomes and the PLC in rat-mouse cell syncytia suggest that the pathway(s) of protein intermixing with respect to the two organelles may be different.

Animals↗

PubMedDate not supplied

Involvement of lysosome-like particles in the metabolism of endogenous myocardial triglycerides during ischemia/reperfusion. Uptake and degradation of triglycerides by lysosomes isolated from rat heart.

The hormonal regulation and enzymatic basis of endogenous lipolysis in heart are not yet completely elucidated. The lysosomal fraction from rat heart appeared to be markedly enriched in triglycerides and a significant reduction in triglycerides in this fraction was found after prolonged perfusion or stimulation of lipolysis with glucagon. The enhanced rate of lipolysis, measured as glycerol release from the isolated perfused rat heart, was abolished 10-15 min after continuous glucagon administration. Omission of glucagon for another 60 min restored the ability of glucagon to stimulate lipolysis, indicating the limited availability of endogenous triglycerides and the presence of a transfer-system for triglycerides from a non-metabolically active pool to a metabolically active pool. The enhanced lipolysis induced by low-flow ischemia was found to be inhibited by the lysosomotropic agent methylamine (5 mM). Methylamine-perfusion during low-flow ischemia was accompanied by an increased recovery of myocardial triglycerides in the lysosomal fraction. The possible role of lysosome-like particles in myocardial triglyceride homeostasis was further investigated by studying the kinetics of uptake and degradation of labeled triglycerides by membrane-particles recovered in the subcellular fraction enriched with lysosomal marker enzymes. It appeared that isolated lysosomal membranes take up added triglycerides at an average rate of 30 nmoles/min/g protein. The bulk of these triglycerides taken up is stored whereas 20% is degraded to diglycerides and free fatty acids. More than 90% of the free fatty acids formed were released from the lysosomes into the supernatant. The uptake and degradation of triglyceride-filled liposomes by isolated myocardial lysosomes was inhibited during incubation with methylamine (5 mM). On the other hand, a lowering of pH during in vitro incubation increased the rate of uptake and degradation of added triglycerides by isolated lysosomes. These results indicate that lysosomes or lysosome-like particles are involved in the enhanced lipolysis during myocardial ischemia.

Acid-Base Equilibrium↗

PubMed1992

Drug-induced lysosomal disorders in laboratory animals: new substances acting on lysosomes.

Several substances with lysosomotropic activity were investigated in toxicological studies. AR-L 115 BS (sulmazol, a cardiotonic agent) was tested on beagle dogs; HX-CH 44 BS (a beta-blocker) and SX-AB 1316 SE (an antithrombotic agent) were tested on rats, and AF-CX 1325 XX (an antiepileptic agent) was tested on both rats and beagle dogs. All organ systems were examined morphologically by light and/or electron microscopy. When an increase in the number of lysosomes occurred this was confirmed by the pigment scheme according to Krutsay (1971) as well as by the detection of acid phosphatase and compared with earlier histochemical results. At higher dosages, all substances caused very marked proliferation of lysosomes in the liver and/or kidneys. HX-CH 44 BS also caused such proliferation in striated muscles and in the lungs. A brown discolouration of the kidneys was found with sulmazol and AF-CX 1325 XX. This finding corresponded to the microscopically detectable occurrence of numerous lipofuscin granules. The reticulum cells in the lymph nodes of dogs were also affected by AF-CX 1325 XX. It is concluded that the proliferation of lysosomes in various organs after administration of the above-mentioned substances is due to an excess of substance. The increased substance in the body is then stored in the lysosomes. With HX-CH 44 BS, lysosomal autodigestion of mitochondria in the skeletal musculature and in the alveolar macrophages of the lungs was found. The selective lysosomal incorporation of mitochondria has not been described up to now and in our opinion, this constitutes a special feature. The results otherwise largely correspond to those already described in the literature. Systemic phospholipidosis such as occurs with some other substances was not detectable. The incorporation of the substance causes several types of lysosomal inclusion. Uptake of the substance in lysosomes either leads to overt autodigestion of organelles such as mitochondria (HX-CH 44 BS) or peroxisomes or to residual lysosomes of dense structure which histochemically resemble lipofuscin. SX-AB 1316 SE serves as an example of a substance which is stored directly by lysosomes in crystalline form. Above all, in the liver the substance is taken up not only by the sinusoidal stellate cells but also by hepatocytes.

Adrenergic beta-Antagonists↗

PubMed1993

Effect of reactive oxygen species on lysosomal membrane integrity. A study on a lysosomal fraction.

Using a lysosome-enriched "light mitochondrial" fraction of a rat liver homogenate, the effects of the reactive oxygen species hydrogen peroxide, superoxide- and hydroxyl radicals were determined. Alterations in the intralysosomal pH and the release of a lysosomal marker enzyme, N-acetyl-glucosaminidase, were used as indicators of changes in the lysosomal membrane integrity. Lipid peroxidation of the fraction was assayed by TBARS measurement. Neither superoxide radicals, generated by hypoxanthine/xanthine oxidase, nor a bolus dose of hydrogen peroxide (0.5-1.5 mM) induced any lysosomal damage. If, however, Fe(III)ADP was included in the superoxide radical-generating system, lysosomal membrane damage was detected, both as an increase in lysosomal pH and as a release of N-acetyl-glucosaminidase, but only after a lag phase of about 7 min. Lipid peroxidation, on the other hand, proceeded gradually. Lysosomes treated with hydrogen peroxide displayed similar dose-dependent alterations, albeit only if both Fe(III)ADP and the reducing amino acid cysteine were added. In the latter system, however, alterations of the lysosomal membrane stability occurred more rapidly, showing a lag phase of only 2 min. Lipid peroxidation, which proceeded faster and displayed no lag phase, levelled out within 10 min. The results indicate that neither superoxide radicals nor hydrogen peroxide are by themselves damaging to lysosomes. Available catalytically active iron in Fe(II) form, however, allows reactions yielding powerful oxidative species--probably hydroxyl radicals formed via Fenton reactions--to take place inducing peroxidation of the lysosomal membranes resulting in dissipation of the proton-gradient and leakage of their enzyme contents.

Animals↗

PubMed1987

An improved procedure for the isolation of lamellar bodies from human lung. Lamellar bodies free of lysosomes contain a spectrum of lysosomal-type hydrolases.

We have recently shown that lamellar body fractions purified from human lung contain a distinct acid alpha-glucosidase distinguishable from lysosomal acid alpha-glucosidase in that it does not cross-react with antibodies raised against the lysosomal enzyme and does not bind to concanavalin A (De Vries, A.C.J., Schram, A.W., Tager, J.M., Batenburg, J.J. and Van Golde, L.M.G. (1985) Biochim. Biophys. Acta 837, 230-238). In order to study the relationship between the non-concanavalin A-binding alpha-glucosidase and lamellar bodies more closely a method was developed for the further purification of the organelles. A purified lamellar body preparation isolated from human lung homogenate by discontinuous sucrose density centrifugation was subjected to gel filtration with Sepharose 4B followed by Percoll density gradient centrifugation, which yielded a lamellar body preparation with a phospholipid phosphorus/protein ratio of 12.57 +/- 0.38 (mumol/mg) (n = 3) as compared to a ratio of 3.34 +/- 0.16 (mumol/mg) (n = 3) in the sucrose density gradient preparation. Concomitantly there was a 3.3 +/- 0.1 (n = 3)-fold enrichment in the content of total acid alpha-glucosidase and a 3.2 +/- 0.1 (n = 3) -fold enrichment of non-concanavalin A-binding acid alpha-glucosidase. The new purification method removes adhering proteins without changing the phospholipid composition. During the successive purification steps the concanavalin A-sensitive and -insensitive alpha-glucosidases remained fully lamellar body fraction associated. Differences between a lysosome-enriched fraction and a lamellar body preparation at varying stages of purification with respect to the ratio between soluble acid hydrolases and the membrane-associated lysosomal enzyme glucocerebrosidase indicate that the purified lamellar bodies were not contaminated with lysosomes. The absence of lysosomes in the purified lamellar body fraction was confirmed by experiments with the weak base glycyl-L-phenylalanine-beta-naphthylamide, which is an artificial substrate for the lysosomal enzyme cathepsin C and brings about lysis of lysosomes. Morphological examination by electron microscopy endorses the absence of contaminating vesicles and organelles and showed a structural integrity of the lamellar bodies in the final preparation. The improved isolation procedure strongly suggests that the concanavalin A-insensitive acid alpha-glucosidase is endogenous to lamellar bodies and supports our earlier idea that it can be used as a lamellar body-specific marker enzyme. In addition, the experiments show that lamellar bodies free of lysosomes contain a spectrum of lysosomal-type enzymes.

Cell Fractionation↗

PubMed1976

Physicochemical characteristics of the glycosaminoglycan-lysosomal enzyme interaction in vitro. A model of control of leucocytic lysosomal activity.

1. The activities of 30 different lysosomal enzymes were determined in vitro in the presence of the sulphated glycosaminoglycans, heparin and chondroitin sulphate, all the enzymes being measured on a density-gradient-purified lysosomal fraction. 2. Each enzyme was studied as a function of the pH of the incubation medium. In general the presence of sulphated glycosaminoglycans induced a strong pH-dependent inhibition of lysosomal enzymes at pH values lower than 5.0, with full activity at higher pH values. However, in the particular case of lysozyme and phospholipase A2 the heparin-induced inhibition was maintained in the pH range 4.0-7.0. 3. For certain enzymes, such as acid beta-glycerophosphatase, alpha-galactosidase, acid lipase, lysozyme and phospholipase A2, the pH-dependent behaviour obtained in the presence of heparin was quite different to that obtained with chondroitin sulphate, suggesting the existence of physicochemical characteristic factors playing a role in the intermolecular interaction for each of the sulphated glycosaminoglycans studied. 4. Except in the particular case of peroxidase activity, in all other lysosomal enzymes measured the glycosaminoglycan-enzyme complex formation was a temperature-and time-independent phenomenon. 5. The effects of the ionic strength and pH on this intermolecular interaction reinforce the concept of an electrostatic reversible interaction between anionic groups of the glycosaminoglycans and cationic groups on the enzyme molecule. 6. As leucocytic primary lysosomes have a very acid intragranular pH and large amounts of chondroitin sulphate, we propose that this glycosaminoglycan might act as molecular regulator of leucocytic activity, by inhibiting lysosomal enzymes when the intragranular pH is below the pI of lysosomal enzymes. This fact, plus the intravacuolar pH changes described during the phagocytic process, might explain the unresponsiveness of lysosomal enzymes against each other existing in primary lysosomes as well as its full activation at pH values occurring in secondary lysosomes during the phagocytic process.

Acid Phosphatase↗