Lysis Of Lysosomes Research Articles

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 α-glucosidase distinguishable from lysosomal acid α-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 α-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 (μmol/mg) ( n = 3) as compared to a ratio of 3.34 ± 0.16 (μmol/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 α-glucosidase and a 3.2 ± 0.1 ( n = 3) -fold enrichment of non-concanavalin A-binding acid α-glucosidase. The new purification method removes adhering proteins without changing the phospholipid composition. During the successive purification steps the concanavalin A-sensitive and -insensitive α-gluco-sidases 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-β-naph-thylamide, 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 α-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.

Transport and processing of endocytosed lysosomal alpha-glucosidase in cultured human skin fibroblasts.

In the present study we have characterised the molecular products that arise from processing of a precursor form of alpha-glucosidase isolated from urine after endocytosis at 37 degrees C by cultured human skin fibroblasts. The urinary precursor (Mr 110 000) was processed to forms with Mr of 100 000, 80 000 and 74 000. These forms were approximately 4000 Da larger than the corresponding forms of endogenously synthesized alpha-glucosidase. Digestion of the different forms of the enzyme with endoglycosidase F showed that the differences in apparent molecular mass between the exogenous and corresponding endogenous forms were due to difference in glycosylation. Intracellular transport of endocytosed alpha-glucosidase was followed by incubating fibroblast homogenates with glycyl-L-phenylalanine-beta-naphthylamide (Gly-Phe-NH-Nap), which leads to specific lysis of lysosomes. Transport to the lysosomes was a fast process: within 45 min after endocytosis more than 50% of the enzyme was present in the lysosome. The first step in the processing of endocytosed alpha-glucosidase started in a Gly-Phe-NH-Nap-insensitive (prelysosomal) compartment, but further processing of the enzyme to lower-Mr forms was coupled to transport to the lysosomes. Processing of alpha-glucosidase after uptake at 20 degrees C was also studied. At this temperature the enzyme accumulated in an organelle with a low buoyant density, presumably the endosome; this compartment appeared to be heterogeneous, ranging in density from 1.04 g/ml to 1.08 g/ml. Under these conditions only the first step in the processing of the enzyme occurred. It is concluded that endocytosed enzyme is processed more rapidly than endogenously synthesized enzyme owing to the fact that endocytosed enzyme is transported more rapidly to the lysosomes. Furthermore, processing may start in a prelysosomal organelle.

Effect of glycyrrhizin on stability of lysosomes in the rat arterial wall.

Studies were made on the effect of phospholipase A2 on the stability of arterial wall lysosomes, and the influence of glycyrrhizin on this effect. Lysosomal phospholipase A2 activity was found to be increased in the aorta of hypercholesterolemic rats. Treatment of the lysosomal fraction of the arterial wall with venom phospholipase A2 resulted in release of acid phosphatase, and addition of glycyrrhizin (0.1 mM) inhibited this release. Lysosomal phospholipase A2 in the arterial wall was also inhibited dose-dependently by glycyrrhizin. These results suggest that lysis of lysosomes was due to increase in phospholipase A2 activity, and that glycyrrhizin stabilized the lysosomes by inhibiting phospholipase A2 activity.

Biosynthesis and intracellular transport of α‐glucosidase and cathepsin D in normal and mutant human fibroblasts

In order to study the intracellular localization of the proteolytic processing steps in the maturation of alpha-glucosidase and cathepsin D in cultured human skin fibroblasts we have used incubation with glycyl-L-phenylalanine-beta-naphthylamide (Gly-Phe-NH-Nap) as described by Jadot et al. [Jadot, M., Colmant, C., Wattiaux-de Coninck, S. & Wattiaux, R. (1984) Biochem. J. 219,965-970] for the specific lysis of lysosomes. When a homogenate of fibroblasts was incubated for 20 min with 0.5 mM Gly-Phe-NH-Nap, a substrate for the lysosomal enzyme cathepsin C, the latency of the lysosomal enzymes alpha-glucosidase and beta-hexosaminidase decreased from 75% to 10% and their sedimentability from 75% to 20-30%. In contrast, treatment with Gly-Phe-NH-Nap had no significant effect on the latency of galactosyltransferase, a marker for the Golgi apparatus, and on the sedimentability of glutamate dehydrogenase and catalase, markers for mitochondria and peroxisomes, respectively. The maturation of alpha-glucosidase and cathepsin D in fibroblasts was studied by pulse-labelling with [35S]methionine, immunoprecipitation, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and fluorography. When homogenates of labelled fibroblasts were incubated with Gly-Phe-NH-Nap prior to immunoprecipitation, 70-80% of all proteolytically processed forms of metabolically labelled alpha-glucosidase and cathepsin D was recovered in the supernatant. The earliest proteolytic processing steps in the maturation of alpha-glucosidase and cathepsin D appeared to be coupled to their transport to the lysosomes. Although both enzymes are transported via the mannose-6-phosphate-specific transport system, the velocity with which they arrived in the lysosomes was consistently different. Whereas newly synthesized cathepsin D was found in the lysosomes 1 h after synthesis, alpha-glucosidase was detected only after 2-4 h. When a pulse-chase experiment was carried out in the presence of 10 mM NH4Cl there was a complete inhibition of the transport of cathepsin D and a partial inhibition of that of alpha-glucosidase to the lysosomes. Leupeptin, an inhibitor of lysosomal thiol proteinases, had no effect on the transport of labelled alpha-glucosidase to the lysosomes. However, the early processing steps in which the 110-kDa precursor is converted to the 95-kDa intermediate form of the enzyme were delayed, a transient 105-kDa form was observed and the conversion of the 95-kDa intermediate form to the 76-kDa mature form of the enzyme was completely inhibited.(ABSTRACT TRUNCATED AT 400 WORDS)

Changes in muscle subcellular fractions of baltic herring (Clupea harengus membras) during cold and frozen storage

AbstractThe autolytic enzymic degradation of the muscle of Baltic herring (Clupea harengus membras) was studied by investigating the muscle fractions after two different types of storage commonly used in fish handling, i.e. freezing and storage at ‐ 30°C, and storage at +5°C, comparing them with the muscle fractions of fresh fish. Nuclear, mitochondrial, lysosomal and microsomal as well as soluble fractions were studied by assaying the activities of lysosomal cathepsin D and mitochondrial cytochrome c oxidase. The morphology of sedimented fractions as revealed by electron microscopy compared with the enzymic results. After storage, the marker enzymes were detected in the more slowly sedimenting fractions. Freezing and thawing also resulted in the lysis of mitochondria and lysosomes and in a loose, disorganised muscle structure.

Protoporphyrin-induced photodamage to mitochondria and lysosomes from rat liver

Preparations of mitochondria and lysosomes from rat liver were irradiated (340–380 nm) with light doses up to 80 kJ/m 2 in the presence of 180 nmol/1 protoporphyrin. ADP-stimulated respiration and succinate oxidation were inhibited before there was any significant release or inhibition of lysosomal enzymes. The results suggest that, rather than lysosomal lysis, damage to mitochondria may be the primary event in erythropoietic protoporphyria.

An improved method for the preparation of undegraded polysomes and active messenger RNA from immature chicken erythrocytes

A simple method for the large-scale isolation of undegraded polysomes and active mRNA from immature hen erythrocytes is described. The method is based on the removal of white cells from heparinized blood by adsorption on sulfoethyl- or sulfopropyl-Sephadex. Red cells thus purified can be stored frozen and lysed in the presence of nonionic detergents. The yield of polysomes is about twice that obtained by the usual mild lysis procedures designed to prevent lysis of lysosomes from contaminating white cells.

The permeability of the lysosomal membrane to small ions

The permeability of the lysosomal membrane to small anions and cations was studied at 37°C and pH 7.0 in a lysosomal-mitochondrial fraction isolated from the liver of untreated rats. The extent of osmotic lysis following ion influx was used as a measure of ion permeancy. In order to preserve electroneutrality, anion influx was coupled to an influx of K + in the presence of valinomycin, and cation influx was coupled to an efflux of H + using the protonophore 3-tert- butyl-5,2′-dichloro-4′-nitrosalicilylanilide . Lysosomal lysis was monitored by observing the loss of latency of two lysosomal hydrolases. The order of permeability of the lysosomal membrane to anions was found to be SCN − > I − > CH 3COO − > Cl − ≈ HCO − 3 ≈ P i > SO 4 2− and that to cations Cs + > K + > Na + > H + . These orders are largely in agreement with the lyotropic series of anions and cations. The implications of these findings for the mechanism by means of which a low intralysosomal pH is produced and maintained are discussed.

Evidence that Peroxidation of Lysosomal Membranes Is Initiated by Hydroxyl Free Radicals Produced during Flavin Enzyme Activity

Since evidence had been published that lysosomes are disrupted by a free radical-like agent produced during the oxidation of NADPH by liver microsomes, the nature of this free radical was investigated. The following data are presented. 1. The oxidation of NADPH by liver microsomes in the presence of ADP and Fe3+ produced a factor with properties of a free radical which caused the lysis of lysosomes. The addition of superoxide dismutase enhanced the lysis. 2. Lysis of lysosomes also was mediated by the oxidation of NADPH by a partially purified NADPH-cytochrome P450 reductase. 3. The oxidation of xanthine by xanthine oxidase in the presence of ADP and Fe3+ produced a radical capable of causing lysosomal lysis. Here again superoxide dismutase caused an enhancement of this lysis. 4. It was demonstrated that the superoxide free radical per se was not the agent causing lysis. Evidence is presented that the free radical causing the lysis of the lysosomes is the hydroxyl free radical (OH·). 5. During lysosomal lysis by the OH· radical, peroxidation of the lysosomal lipids was observed as assayed by the formation of malondialdehyde. 6. The mechanisms whereby superoxide and hydroxyl free radicals may be produced and interact with lysosomal membranes are discussed.

Preservation of structural integrity of liver lysosomes and membrane-stabilizing action of anti-inflammatory drugs, catecholamines and cyclic adenosine monophosphate in isotonic salt media

Since the first description of lysosomes, most studies with these cytoplasmic organelles have been conducted in sucrose solutions containing little or no inorganic ions. Lysosomal enzymes have been characterized by their structure-linked latency in sucrose solutions of very low ionic strength. The data in this report illustrate that isotonic concentrations of certain inorganic ions can replace sucrose for the purpose of retarding osmotic lysis of liver lysosomes and thus preserving the latent properties of lysosomal enzymes. Acid phosphatase, β-glucuronidase, β-galactosidase and aryl sulfatase were the lysosomal marker enzymes measured. Iso-osmotic concentrations of potassium chloride and Hanks' Balanced Salt Solution were found to be suitable substitutes for sucrose in maintaining the structural integrity of lysosomes at neutral pH and 37°. Certain steroidal and non-steroidal anti-inflammatory drugs, such as hydrocortisone, chloroquine, acetylsalicylic acid, phenylbutazone, indomethacin and flufenamic acid, inhibited the release of several enzymes from lysosomes incubated in isotonic salt media. Further, the catecholamines, norepinephrine, epinephrine and isoproterenol, were found to inhibit lysosomal enzyme release in salt media. Similar lysosome membrane-stabilizing actions were obtained with cyclic 3',5'-adenosine monophosphate and its N 6, O 2′-dibutyryl analog. The data reveal that sucrose can be replaced by certain salt solutions for the purpose of maintaining the integrity of lysosomes or demonstrating drug-induced lysosome membrane stabilization in vitro.

Changes in lipid composition of tritosomes during lysis

Membrane phospholipids and free fatty acids were analyzed after lysis of lysosomes which had engulfed Triton WR 1339 (tritosomes). Significant elevations of lysophosphatidyl choline and lysophosphatidyl ethanolamine were seen. Phosphatidyl choline, phosphatidyl ethanolamine and sphingomyelin decreased significantly. Triglyceride also fell. The released free fatty acids paralleled closely the liberation of β-glucuronidase from the tritosomes. Thus, lipases hydrolyzed endogenous phospholipids and neutral lipids of tritosomes during incubation at 37°C at pH 5.0 to produce significant elevations of lysophospholipids and free fatty acids. The known surface-active properties of these latter compounds raise the possibility of their participation in the process of lysis of the tritosomes.

Progesterone-induced permeability changes in rat liver lysosomes.

RAT liver lysosomes have been shown1,2 to act as “osmotic systems” and to be stable in 0.25 M sucrose solution, presumably as the lysosomal membrane was not permeable to sucrose. Suspension in 0.125 M sodium chloride led to a transient osmotic protection and graded lysis, where the rate-limiting step was presumed to be entry of the sodium chloride. The effects of fat-soluble vitamins and steroids on lysosomes, in vitro, were extensively studied3,4. De Duve et al.3 suggested that compounds causing lysis of lysosomes may act by initiating a sequence of events involving altered permeability to solutes, osmotic swelling and rupture. We report here experiments which support the hypothesis that progesterone-induced lysis of lysosomes suspended in 0.25 M sucrose is due to an increased permeability of the membrane to sucrose leading to osmotic rupture.