In vivo interaction between mouse liver lysosomes and chloroquine

Particles sedimenting at 27,000 g X 10 min (MLCQ) were separated from liver homogenates of mice injected with chloroquine (CQ). The MLCQ contained most of the drug recovered in the organ as well as 50% of the liver aryl sulphatase activity. The release of CQ from MLCQ was studied in some physicochemical conditions, and in the presence of various agents known to modify membrane composition and stability. At pH 7.4, the equilibrium between free and bound CQ depended on the dilution of the MLCQ, and the time to reach equilibrium was strongly influenced by the temperature of incubation. Several agents causing membrane disruption and lysosomal enzyme leakage, such as osmotic shock, sonication and digitonin, had little effect on the CQ release. Acid and alkaline buffers, 0.55 M KCl and 0.1% Triton X-100 caused, instead, the immediate release of most of the bound CQ. Concentrations of digitonin causing the release of aryl sulphatase activity had little effect on bound CQ, suggesting that the drug is retained in lysosomes by forces and/or structures different in nature from those retaining most of the lysosomal enzyme activity. We think that the CQ trapped in lysosomes is bound to high affinity sites in membranous structures which are particularly altered by agents known to extract peripheral proteins from biological membranes or to change the conformation of molecular structures.     

1H-NMR effects in chloroquine-biopolymer binding interactions.

Chloroquine (CQ), an antimalarial and anti-inflammatory drug, is known to concentrate within lysosomes. 1H-NMR studies were conducted using the resonances of CQ itself during binding interactions with various polymers and proteins including lysosome fractions isolated from rodent livers by tritosome technique. Albumin, butyrylcholinesterase, and high molecular weight DNA interact with CQ, producing marked line-width changes that correlate with effective molecular weight. Triton WR-1339 and sucrose, probable contaminants of the lysosomal materials isolated, produced essentially no effect beyond a viscosity component. Lysosomal matrix and membrane fractions exhibited relatively weak interactions, membranes being the more tenacious toward CQ. Estimated binding constants are too small to permit explanation of CQ uptake in terms of protein affinity. The evidence is more consistent with a proton-pump trapping model proposed by de Duve et al.     

Chloroquine-mediated radiosensitization is due to the destabilization of the lysosomal membrane and subsequent induction of cell death by necrosis

The anti-malarial drug chloroquine (CQ) is also thought to be a potential radiation sensitizer. To gain a better understanding of how the lysomotropic CQ can potentiate the effects of ionizing radiation, we investigated the effects of CQ on lysosomal and mitochondrial membrane stability, the subcellular localization of ceramide, plasma membrane permeability, and the mode of cell death in response to irradiation. We found that CQ accumulated in the lysosomes and thus lysosomal volumes increased. As a result, both the lysosomal and plasma membranes were destabilized. After 7 Gy irradiation, most ceramide was associated with the lysosomes in the cells treated with CQ but not in the CQ-untreated control. The elevated levels of ceramide in the lysosomes of the CQ-treated cells appeared to further destabilize the lysosomal and plasma membranes of the cell. Both CQ-treated and -untreated cells had approximately the same rate of cell death by apoptosis after 7 Gy irradiation (P > 0.05, ns). However, in contrast to the CQ-untreated control, the CQ-treated cells underwent massive cell death by necrosis at 24-48 h after irradiation (P < 0.05). Taken together, our data support the idea that the increase in cytotoxic effects by the combination of CQ and radiation is due to radiation-mediated apoptosis and CQ-mediated necrosis.     

Cellular uptake of chloroquine is dependent on binding to ferriprotoporphyrin IX and is independent of NHE activity in Plasmodium falciparum

Here we provide definitive evidence that chloroquine (CQ) uptake in Plasmodium falciparum is determined by binding to ferriprotoporphyrin IX (FPIX). Specific proteinase inhibitors that block the degradation of hemoglobin and stop the generation of FPIX also inhibit CQ uptake. Food vacuole enzymes can generate cell-free binding, using human hemoglobin as a substrate. This binding accounts for CQ uptake into intact cells and is subject to identical inhibitor specificity. Inhibition of CQ uptake by amiloride derivatives occurs because of inhibition of CQ-FPIX binding rather than inhibition of the Na+/H+ exchanger (NHE). Inhibition of parasite NHE using a sodium-free medium does not inhibit CQ uptake nor does it alter the ability of amilorides to inhibit uptake. CQ resistance is characterized by a reduced affinity of CQ-FPIX binding that is reversible by verapamil. Diverse compounds that are known to disrupt lysosomal pH can mimic the verapamil effect. These effects are seen in sodium-free medium and are not due to stimulation of the NHE. We propose that these compounds increase CQ accumulation and overcome CQ resistance by increasing the pH of lysosomes and endosomes, thereby causing an increased affinity of binding of CQ to FPIX.     

Isolation of highly purified rat liver lysosomal membranes using two Percoll gradients

Normal rat liver lysosomal membranes in the form of membrane vesicles have been purified using Percoll density gradient centrifugation. Lysosomes (density = 1.111) were purified approximately 63 +/- 12-fold (mean +/- standard deviation, n = 5) using a gradient of Percoll made isotonic with sucrose and buffered to pH 7.0. These lysosomes were then exposed to 10 mM methionine methyl ester, pH 7.0, the uptake of which resulted in swelling and breakage of the lysosomes with subsequent vesicle formation. These vesicles (density = 1.056) were further separated from residual mitochondrial and plasma membrane enzyme activities using a second Percoll density gradient. Marker enzyme analysis and electron microscopy indicated that the lysosomal membranes were essentially free of both beta-hexosaminidase, a soluble lysosomal enzyme, and contaminating organelles. The specific activity of lysosomal ATPase in the lysosomal membranes was fourfold greater than in the intact lysosomes.     

Distribution of electric charges and concanavalin A binding sites on autophagic vacuoles and lysosomes in mouse hepatocytes

The distributions of electric charges and Concanavalin A binding sites in autophagic vacuoles and lysosomes in mouse hepatocytes were studied by utilizing a frozen ultrathin section labeling method with cationized ferritin (CF) or anionized ferritin and ferritin-conjugated Concanavalin A (Con A-F) as visual probes. Our observations revealed that the inner surface of the autophagic vacuole membrane has more anionic sites (CF binding) than other organelle membranes. This suggests that if the limiting membranes of autophagic vacuoles originate from preexisting membranes, such membranes must undergo structural and compositional alternation during the formation of the autophagic vacuoles. In contrast to CF, Con A-F showed no distinct binding to the membranes of autophagic vacuoles, but the contents of vacuoles displayed varying Con A-F binding, depending on the stage of the autophagic process. Increased binding was seen in more mature autophagic vacuoles. Since lysosomes showed a preferential accumulation of Con A-F particles, molecules with Con A-F binding sites in autophagic vacuoles may be of lysosomal origin. Con A-F distribution varied from lysosome to lysosome in the same cell, indicating heterogeneity of lysosomal contents. These results suggest that ferritin-conjugated lectin labeling methods applied to frozen, ultrathin section are a useful new approach in analyzing the natural history of autophagic vacuoles and the heterogeneity of lysosomes.     

Purification and characterization of dipeptidyl peptidase IV in rat liver lysosomal membranes.

Dipeptidyl peptidase IV (m-DPP IV) in rat liver lysosomal membranes was purified about 50-fold over the lysosomal membranes with 38% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence and in the absence of SDS. The enzyme amounts to about 3% of lysosomal membrane protein constituents. The purification procedures included: extraction of lysosomal membranes by Triton X-100, WGA-Sepharose affinity chromatography, hydroxylapatite chromatography, ion exchange chromatography, and preparative polyacrylamide gel electrophoresis. The enzyme (M(r) 240,000) is composed of two identical subunits with an apparent molecular weight of 110,000. The enzyme contains about 12.4% carbohydrate and the carbohydrate moiety was composed of mannose, galactose, fucose, N-acetylglucosamine, and neuraminic acid in a molar ratio of 14:17:2:24:11. Susceptibility to neuraminidase and immunoreactivity of the enzyme in intact tritosomes were examined to study the topology of the enzyme in tritosomal membranes. Neuraminidase susceptibility and immunoreactivity of the enzyme were not observed in the intact tritosomes until the tritosomes had been disrupted by osmotic shock. This result indicated that both the oligosaccharide chains and the main protein portion of the enzyme are on the inside surface of the tritosomal membranes. Subcellular localization of DPP IV was determined by means of enzyme immunoassay, which indicated that bile canalicular membranes and lysosomal membranes are the major sites of localization, and DPP IV activity in lysosomes was separated into a membrane bound form (60%) and a soluble form (40%). Immunoelectron microscopy clearly confirmed that DPP IV occurs not only in the bile canalicular domain but also in the lysosomes of rat liver.     

Loss of membrane cholesterol influences lysosomal permeability to potassium ions and protons

Cholesterol is an essential component of lysosomal membranes. In this study, we investigated the effects of membrane cholesterol on the permeability of rat liver lysosomes to K⁺ and H⁺, and the organelle stability. Through the measurements of lysosomal β-hexosaminidase free activity, membrane potential, membrane fluidity, intra-lysosomal pH, and lysosomal proton leakage, we established that methyl-β-cyclodextrin (MβCD)-produced loss of membrane cholesterol could increase the lysosomal permeability to both potassium ions and protons, and fluidize the lysosomal membranes. As a result, potassium ions entered the lysosomes through K⁺/H⁺ exchange, which produced osmotic imbalance across the membranes and osmotically destabilized the lysosomes. In addition, treatment of the lysosomes with MβCD caused leakage of the lysosomal protons and raised the intra-lysosomal pH. The results indicate that membrane cholesterol plays important roles in the maintenance of the lysosomal limited permeability to K⁺ and H⁺. Loss of this membrane sterol is critical for the organelle acidification and stability.     

Characterization of the membrane proteins of rat liver lysosomes. Composition, enzyme activities and turnover.

Lysosomes prepared from the livers of untreated rats and from the livers of rats injected with either Triton WR-1339 or dextran yielded membranes that were similar in both polypeptide composition and activities of ATPase and acid 5'-nucleotidase. The administration of Triton WR-1339 (and dextran) resulted in an increase in ATPase activity of liver homogenates that was associated with a parallel increase in the ATPase activity of the lysosomal membrane. On the other hand, plasma membranes appear to be different from lysosomal membranes with respect to polypeptide composition and enzyme activities. The ATPase activity of lysosomal membranes is not affected by ouabain and suramin, inhibitors of the plasma-membrane ATPase. The plasma-membrane alkaline 5'-nucleotidase has little activity at acid pH. Pulse-labelling of lysosomal membranes with [3H]fucose and with [3H]- and [14C]-leucine occurred rapidly, faster than labelling of plasma membranes. The labelling kinetics indicate that lysosomal membranes may be assembled independently of plasma membranes. These data suggest that, in liver, little bulk transport of plasma membrane to lysosomes takes place, and lysosomal-membrane proteins may not be derived from those of plasma membranes.     

Intracellular localization of p40, a protein identified in a preparation of lysosomal membranes

Unlike lysosomal soluble proteins, few lysosomal membrane proteins have been identified. Rat liver lysosomes were purified by centrifugation on a Nycodenz density gradient. The most hydrophobic proteins were extracted from the lysosome membrane preparation and were identified by MS. We focused our attention on a protein of approx. 40 kDa, p40, which contains seven to ten putative transmembrane domains and four lysosomal consensus sorting motifs in its sequence. Knowing that preparations of lysosomes obtained by centrifugation always contain contaminant membranes, we combined biochemical and morphological methods to analyse the subcellular localization of p40. The results of subcellular fractionation of mouse liver homogenates validate the lysosomal residence of p40. In particular, a density shift of lysosomes induced by Triton WR-1339 similarly affected the distributions of p40 and beta-galactosidase, a lysosomal marker protein. We confirmed by fluorescence microscopy on eukaryotic cells transfected with p40 or p40-GFP (green fluorescent protein) constructs that p40 is localized in lysosomes. A first molecular characterization of p40 in transfected Cos-7 cells revealed that it is an unglycosylated protein tightly associated with membranes. Taken together, our results strongly support the hypothesis that p40 is an authentic lysosomal membrane protein.     

On the entry of semliki forest virus into BHK-21 cells

The pathway by which semliki forest virus (SFV), a membrane-containing animal virus, enters BHK-21 cells was studied morphologically and biochemically. After attaching to the cell surface, the majority of viruses was rapidly trapped into coated pits, internalized by endocytosis in coated vesicles, and sequestered into intracellular vacuoles and lysosomes. Direct penetration of viruses through the plasma membrane was never observed. To assess the possible involvement of lysosomes in the release of the genome into the cytoplasm, the effect of five lysosomotropic agents, known to increase the lysosomal pH, was tested. All of these agents inhibited SFV infectivity and one, chloroquine (the agent studied in most detail), inhibited a very early step in the infection but had no effect on binding, endocytosis, or intracellular distribution of SFV. Thus, the inhibitory effect was concluded to be either on penetration of the nucelocapsid into the cytoplasm or on uncoating of the viral RNA. Possible mechanisms for the penetration of the genome into the cytoplasm were studied in vitro, using phospholipids-cholesterol liposomes and isolated SFV. When the pH was 6.0 or lower, efficient fusion of the viral membranes and the liposomal membranes occurred, resulting in the transfer of the nucleocapsid into the liposomes. Infection of cells could also be induced by brief low pH treatment of cells with bound SFV under conditions where the normal infection route was blocked. The results suggest that the penetration of the viral genome into the cytosol takes place intracellularly through fusion between the limiting membrane of intracellular vacuoles and the membrane of viruses contained within them. The low pH required for fusion together with the inhibitory effect of lysosomotropic agents implicate lysosomes, or other intracellular vacuoles with sufficiently low pH, as the main sites of penetration.     

Detection and characterization of a nucleoside transport system in human fibroblast lysosomes

Lysosomes contain enzymatic activities capable of degrading nucleic acids to their constituent nucleosides, but the manner by which these degradation products are released from the lysosome is unknown. To investigate this process, human fibroblast lysosomes, purified on Percoll density gradients, were incubated with [3H]adenosine at pH 7.0, and the amount of adenosine taken up by the lysosomes was measured. Adenosine uptake by fibroblast lysosomes attained a steady state by 12 min at 37 degrees C and was unaffected by the presence of 2 mM MgATP or changes in pH from 5.0 to 8.0. An Arrhenius plot was linear with an activation energy of 12.9 kcal/mol and a Q10 of 2.0. Lysosomal adenosine uptake is saturable, displaying a Km of 9 mM at pH 7.0 and 37 degrees C. Various nucleosides and the nucleobase, 6-dimethylaminopurine, strongly inhibit lysosomal adenosine uptake, whereas neither D-ribose or nucleotide monophosphates have any significant effect upon lysosomal adenosine uptake. On a molar basis, purines are recognized more strongly than pyrimidines. Changing the nature of the nucleoside sugar from ribose to arabinose or deoxyribose has little effect on reactivity with this transport system. The known plasma membrane nucleoside transport inhibitors, dipyridamole and nitrobenzylthioinosine, inhibit lysosomal nucleoside transport at relatively low concentrations (25 microM) relative to the Km of 9 mM for lysosomal adenosine uptake. The half-times of [3H]inosine and [3H]uridine efflux from fibroblast lysosomes ranged from 6 to 8 min at 37 degrees C. Trans effects were not observed to be associated with either inosine or uridine exodus. In contrast to adenosine uptake, adenine primarily enters fibroblast lysosomes by a route not saturable by high concentrations of various nucleosides. In conclusion, the saturability of lysosomal adenosine uptake and its specific, competitive inhibition by other nucleosides indicate the existence of a carrier-mediated transport system for nucleosides within fibroblast lysosomal membranes.     

Proliferin secreted by cultured cells binds to mannose 6-phosphate receptors

Proliferin is a prolactin-related glycoprotein secreted by proliferating mouse cell lines and by mouse placenta. In an attempt to identify target sites for proliferin action, we looked for proliferin receptors in murine fetal and maternal tissues during pregnancy using proliferin purified from the conditioned medium of a constructed Chinese hamster ovary cell line carrying amplified copies of proliferin cDNA. Purified proliferin bound to membrane preparations from fetal or maternal liver and from placenta with a Kd of 1 to 2 nM. The amount of proliferin bound per microgram of membrane protein varied markedly during pregnancy; maximal binding to day 16 fetal liver membranes was approximately 25 times that to liver membranes from adult animals. Binding to fetal and maternal receptors was specifically and completely inhibited by mannose 6-phosphate, with half-maximal inhibition at 10 microM. Furthermore, non-glycosylated proliferin did not inhibit the binding of the glycosylated protein. A approximately 300 Kd proliferin receptor was purified from the liver of pregnant mice using a proliferin affinity column and elution with mannose 6-phosphate. This receptor reacted with antibodies directed against the rat cation-independent mannose 6-phosphate receptor. We conclude that 1) proliferin secreted by cultured cell binds to cation-independent mannose 6-phosphate receptors and therefore may be a lysosomal protein or targeted to lysosomes, and 2) the concentration or activity of mannose 6-phosphate receptors in murine fetal and maternal liver and in placenta is regulated during pregnancy.     

Receptors for gonadotropins and prostaglandins in lysosomes of bovine corpora lutea.

The total mitochondrial fraction of bovine corpus luteum specifically bound [³H]prostaglandin (PG) E₁, [³H] PGF_2α, and ¹²⁵I-labeled human lutropin (hLH) despite very little 5′-nucleotidase activity, a marker for plasma membranes. Since the total mitochondrial fraction isolated by conventional centrifugation techniques contains both mitochondria and lysosomes, it was subfractionated into mitochondria and lysosomes to ascertain the relative contribution of these fractions to the binding. Subfractionation resulted in an enrichment of cytochrome c oxidase (a marker for mitochondria) in mitochondria and of acid phosphatase (a marker for lysosomes) in lysosomes. The lysosomes exhibited little or no contamination with Golgi vesicles, rough endoplasmic reticulum, or peroxisomes as assessed by their appropriate marker enzymes. Subfractionation also re ulted in [³H] PGE₁, [³H] PGF_2α, and ¹²⁵I-labeled hLH binding enrichment with respect to homogenate in lysosomes but not in mitochondria. The lysosomal binding enrichment and recovery were, however, lower than in plasma membranes. The ratios of marker enzyme to binding, an index of organelle contamination, revealed that plasma membrane and lysosomal receptors were intrinsic to these organelles. Freezing and thawing had markedly increased lysosomal binding but had no effect on plasma membrane binding. Exposure to 0.05% Triton X-100 resulted in a greater loss of plasma membrane compared to lysosomal binding. In summary, the above results suggest that lysosomes, but not mitochondria, in addition to plasma membranes, intrinsically contain receptors for PGs and gonadotropins. Furthermore, lysosomes overall contain a greater number of PGs and gonadotropin receptors compared to plasma membranes and these receptors are associated with the membrane but not the contents of lysosomes.     

Glycoprotein catabolism in rat liver. Lysosomal digestion of iodinated asialo-fetuin

(125)I-labelled asialo-fetuin, administered intravenously, rapidly accumulates in rat liver and the radioactivity is subsequently cleared from the liver within 60min. Plasma radioactivity reaches a minimum between 10 and 15 min after injection and rises slightly during the period of liver clearance. Free iodide is the only radioactive compound found in plasma during this latter period. Fractionation of rat liver at 5 and 13min after injection of (125)I-labelled asialo-fetuin supports the hypothesis that asialo-glycoprotein is taken into liver by pinocytosis after binding to the plasma membrane and is then hydrolysed by lysosomal enzymes. At 5min, radioactivity was concentrated 23-fold in a membrane fraction similarly enriched in phosphodiesterase I, a plasma-membrane marker enzyme, whereas at 13min the radioactivity appeared to be localized within lysosomes. Separation of three liver fractions (heavy mitochondrial, light mitochondrial and microsomal) on sucrose gradients revealed the presence of two populations of radioactive particles. One population banded in a region coincident with a lysosomal marker enzyme. The other, more abundant, population of radioactive particles had a density of 1.13 and contained some phosphodiesterase, but very little lysosomal enzyme. These latter particles appear to be pinocytotic vesicles produced after uptake of the asialo-fetuin bound by the plasma membrane. Lysosomal extracts extensively hydrolyse asialo-fetuin during incubation in vitro at pH4.7 and iodotyrosine is completely released from the iodinated glycoprotein. Protein digestion within lysosomes was demonstrated by incubating intact lysosomes containing (125)I-labelled asialo-fetuin in iso-osmotic sucrose, pH7.2. The radioactive hydrolysis product, iodotyrosine, readily passed through the lysosomal membrane and was found in the external medium. These results are not sufficient to account for the presence of free iodide in plasma, but this was explained by the observation that iodotyrosines are deiodinated by microsomal enzymes in the presence of NADPH.     

The isolation of lysosomes from normal rat liver by affinity chromatography

Lysosomes from normal rat liver were isolated by affinity chromatography using Sepharose-bound Ricinus communis agglutinins I + II. Characterization of the lysosomal fraction by marker enzymes showed--compared with the homogenate--an enrichment in: acid phosphatase and arylsulfatase about 30- to 60-fold, the tartrate-sensitive acid phosphatase about 95-fold, whereas beta-D-glucosidase, beta-D-galactosidase and sphingomyelinase showed a much higher enrichment of 170- to 260-fold. Marker enzymes for other cell organelles were not detectable. The phospholipid pattern and optical control with electron microscopy gave further indications that the isolated fractions were very rich in lysosomes. A comparison of the phospholipid compositions of plasma membranes isolated from normal rat liver and membranes from the isolated fractions of lysosomes, showed that they were quite different; in particular bis(monoacylglycero)phosphate, which we found to be a typical lysosomal phospholipid, was absent in plasma membranes.     

Studies on plasma membranes

Summary Plasma membranes and lysosomes were isolated from rat liver and assayed for sialidase activity with gangliosides and sialyllactose as substrates. Plasma membrane and lysosomal activities differed in substrate preference, heat stability, inhibition by Cu²⁺,K _m values and pH dependence. It is concluded that plasma membranes and lysosomes contain different sialidases. Hepatoma plasma membranes also exhibited sialidase activity towards the two substrates.     

ATP-dependent acidification of membrane vesicles isolated from purified rat liver lysosomes. Acidification activity requires phosphate

Membrane vesicles were isolated from purified liver lysosomes of rats treated with Triton WR-1339. In order to preserve ATP-dependent acidification activity, proteolysis of membranes was minimized by adding protease inhibitors and by centrifuging to form dilute bands of vesicles rather than highly concentrated pellets. The membrane vesicle fraction represented about 20% of the total lysosomal protein, 80% of the ATPase activity, and 3% of the solute proteins as marked by N-acetylglucosaminidase. About one-half of the membranes were oriented right side out. The space unavailable to [14C]sucrose corresponded to 3 microliters/mg of membrane protein which indicates that the membranes form vesicles about one-tenth the size of lysosomes. Uptake of either [14C]methylamine or [14C]chloroquine by lysosomal membrane vesicles was ATP-dependent, indicating acidification of the intravesicle space. The acidification activity was inhibited when either 1.5 microM carbonyl cyanide p-trifluoromethoxy-phenylhydrazone, 100 microM dicyclohexylcarbodiimide, or millimolar concentrations of such permeant weak bases as ammonium sulfate and dansyl cadaverine were added. Acidification of lysosomal vesicles by ATP occurred electroneutrally. This acidification activity was not dependent on added salts but was inhibited by the anion transport inhibitors pyridoxal phosphate and diisothiocyanostilbene disulfonic acid, thus suggesting co-transport of protons and anions. Results which indicate that phosphate is the transported anion included (a) ATP-dependent uptake of [32P]phosphate by lysosomal membrane vesicles and (b) stimulation of ATP-dependent acidification of these vesicles by added phosphate. These observations provide further evidence that maintenance of the acid intralysosomal pH necessary for activation of lysosomal hydrolases is due to an ATP-driven proton pump located in the lysosomal membrane.     

Lysosomal membrane proteins do not bind to mannose-6-phosphate-specific receptors

Lysosomal membrane proteins and soluble lysosomal material were isolated from pulse-chase labelled human skin fibroblasts and examined for incorporation of radioactivity and affinity to immobilized mannose-6-phosphate-specific receptors. Incorporation of radioactivity into lysosomal membrane proteins was delayed by about 2 h on average when compared to that of soluble lysosomal proteins. The lack of binding indicates that a mannose-6-phosphate-independent mechanism is responsible for targeting of lysosomal membrane proteins to lysosomes. In contrast to soluble lysosomal proteins, the membrane proteins did not bind to mannose-6-phosphate specific receptors. The delayed appearance of membrane proteins in lysosomes as compared to that of soluble lysosomal proteins suggested that different pathways are utilized by the two classes of lysosomal proteins.     

Influence of membrane physical state on lysosomal potassium ion permeability

Lysosomal permeability to potassium ions is an important property of the organelle. Influence of the membrane physical state on the potassium ion permeability of isolated lysosomes was assessed by measuring the membrane potential with bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol and monitoring the lysosomal proton leakage with p-nitrophenol. The membrane fluidity of lysosomes was modulated by treatment with membrane fluidizer benzyl alcohol and rigidifier cholesteryl hemisuccinate. Changes in the membrane order were examined by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. The measurements of membrane potential and proton leakage demonstrated that the permeability of lysosomes to potassium ions increased with rigidification of their membranes by cholesteryl hemisuccinate treatment at 37 degrees C, and decreased with fluidization of their membranes by benzyl alcohol treatment at 2 degrees C. The changes in ion permeability could be recovered by fluidizing the rigidified membranes and rigidifying the fluidized membranes. The results suggest that the physical states of lysosomal membranes play an important role in the regulation of their K(+) permeability.