Crude Lysosomal Fraction Research Articles

Trehalose causes low-grade lysosomal stress to activate TFEB and the autophagy-lysosome biogenesis response

ABSTRACT The autophagy-lysosome system is an important cellular degradation pathway that recycles dysfunctional organelles and cytotoxic protein aggregates. A decline in this system is pathogenic in many human diseases including neurodegenerative disorders, fatty liver disease, and atherosclerosis. Thus there is intense interest in discovering therapeutics aimed at stimulating the autophagy-lysosome system. Trehalose is a natural disaccharide composed of two glucose molecules linked by a ɑ-1,1-glycosidic bond with the unique ability to induce cellular macroautophagy/autophagy and with reported efficacy on mitigating several diseases where autophagy is dysfunctional. Interestingly, the mechanism by which trehalose induces autophagy is unknown. One suggested mechanism is its ability to activate TFEB (transcription factor EB), the master transcriptional regulator of autophagy-lysosomal biogenesis. Here we describe a potential mechanism involving direct trehalose action on the lysosome. We find trehalose is endocytically taken up by cells and accumulates within the endolysosomal system. This leads to a low-grade lysosomal stress with mild elevation of lysosomal pH, which acts as a potent stimulus for TFEB activation and nuclear translocation. This process appears to involve inactivation of MTORC1, a known negative regulator of TFEB which is sensitive to perturbations in lysosomal pH. Taken together, our data show the trehalose can act as a weak inhibitor of the lysosome which serves as a trigger for TFEB activation. Our work not only sheds light on trehalose action but suggests that mild alternation of lysosomal pH can be a novel method of inducing the autophagy-lysosome system. Abbreviations: ASO: antisense oligonucleotide; AU: arbitrary units; BMDM: bone marrow-derived macrophages; CLFs: crude lysosomal fractions; CTSD: cathepsin D; LAMP: lysosomal associated membrane protein; LIPA/LAL: lipase A, lysosomal acid type; MAP1LC3: microtubule-associated protein 1 light chain 3; MFI: mean fluorescence intensity; MTORC1: mechanistic target of rapamycin kinase complex 1; pMAC: peritoneal macrophages; SLC2A8/GLUT8: solute carrier family 2, (facilitated glucose transporter), member 8; TFEB: transcription factor EB; TMR: tetramethylrhodamine; TREH: trehalase

High perfluorooctanoic acid exposure induces autophagy blockage and disturbs intracellular vesicle fusion in the liver.

Perfluorooctanoic acid (PFOA) has been shown to cause hepatotoxicity and other toxicological effects. Though PPARα activation by PFOA in the liver has been well accepted as an important mechanism of PFOA-induced hepatotoxicity, several pieces of evidence have shown that the hepatotoxic effects of PFOA may not be fully explained by PPARα activation. In this study, we observed autophagosome accumulation in mouse livers as well as HepG2 cells after PFOA exposure. Further in vitro study revealed that the accumulation of autophagosomes was not caused by autophagic flux stimulation. In addition, we observed that PFOA exposure affected the proteolytic activity of HepG2 cells while significant dysfunction of lysosomes was not detected. Quantitative proteomic analysis of crude lysosomal fractions from HepG2 cells treated with PFOA revealed that 54 differentially expressed proteins were related to autophagy or vesicular trafficking and fusion. The proteomic results were further validated in the cells in vitro and livers in vivo after PFOA exposure, which implied potential dysfunction at the late stage of autophagy. However, in HepG2 cells, it seemed that further inhibition of autophagy did not significantly alter the effects of PFOA on cell viability. Although these findings demonstrate that PFOA blocked autophagy and disturbed intracellular vesicle fusion in the liver, the changes in autophagy were observed only at high cytotoxic concentrations of PFOA, suggesting that autophagy may not be a primary target or mode of toxicity. Furthermore, since altered liver autophagy was not observed at concentrations of PFOA associated with human exposures, the relevance of these findings must be questioned.

Atlantic salmon ( Salmo salar) muscle structure integrity and lysosomal cathepsins B and L influenced by dietary n-6 and n-3 fatty acids

This study had two main objectives: first, to evaluate the impact of different types and levels of dietary n-6 and n-3 fatty acids (FAs) on Atlantic salmon muscle structure integrity; second, to highlight a possible role of lysosomes and lysosomal degrading enzymes, cathepsins, in fish muscle structure integrity, in relation to dietary fatty acids. Four groups of Atlantic salmon (90 g starting weight) in fresh water tanks were fed one of four diets containing 23% crude lipids, with 100% of the added oils as either fish oil (FO), rapeseed oil (RO), eicosapentaenoic acid (EPA) enriched-oil or docosahexaenoic acid (DHA) enriched-oil. The RO diet was characterised by low levels of EPA + DHA (10% of total FAs), whereas the EPA and DHA diets were characterised by very high levels of EPA + DHA (>50% of total FAs). Fatty acid composition of the muscle crude lysosomal fraction (CLF) generally reflected the diets. Salmon fed the RO diet presented a muscle CLF FA composition close to the one of the FO group, showing moderate PUFA levels, and comparable cathepsin B and cathepsin L activities, relative gene expression of cathepsin B and cathepsin L in the muscle and rate of myofibre–myofibre detachments post-mortem. Salmon fed the EPA and DHA-enriched-oil diets presented a fairly similar muscle CLF FA composition, but different from the FO and RO groups. In the EPA and DHA groups, the percentage of PUFAs in the muscle CLF, the rate of myofibre–myofibre detachments and the relative gene expression of cathepsin B were higher than in the FO and RO groups. Cathepsin B and cathepsin L total activities in the muscle were however lower in the EPA and DHA groups 0 h post-mortem. Dietary lipids influenced the level of lysosomal degrading enzyme activity cathepsin B and cathepsin L as well as the relative gene expression of cathepsin B. Feeding Atlantic salmon with rapeseed oil and extreme levels of EPA + DHA highlighted the impact of fatty acid composition of the diet on salmon muscle integrity and the complexity of the process involving muscle lysosomes and cathepsins in relation to these dietary fatty acids.

Effects of Mefloquine on the Release of Marker Enzymes from the Rat Liver Crude Lysosomal Fraction

The effects of the antimalarial agent mefloquine on the release of marker enzymes, acid phosphatase and beta glucuronidase, from the rat liver lysosomes in a crude lysosomal preparation were investigated and compared with that of chloroquine whose membrane effects have been well-documented in the literature. At 10 aM, mefloquine decreased significantly the release of marker enzymes when compared to the control, but at the higher concentrations of 100 μM and 500 aM, it markedly accelerated the release of enzymes. This suggested that mefloquine exerted a concentration-dependant biphasic effect of membrane stabilization and labilization. In comparison, chloroquine diminished the release of enzymes over the concentration range of 10-500 μM, that is showing a membrane stabilizing effect similar to other reports. Since serum levels of 1.6-3.2 μM are reported after a weekly dose of mefloquine, the present results suggest that a predominant stabilization effect should prevail under these conditions. However, higher drug concentrations may result in labilization with undesirable consequences.

In vitro inhibition of lysosomal phospholipase A 1 of rat lung by amiodarone and desethylamiodarone

Amiodarone causes phospholipid storage in the lysosomes of various types of lung cell in animals and man. It has been proposed that this is due to its ability to inhibit lysosomal phospholipase A. To investigate this further, a crude lysosomal fraction from rat lung was prepared and phospholipase A was isolated and its positional specificity was determined. Analysis of the products formed after incubation with 2-[1- 14C]oleoylphosphatidylcholine showed that only phospholipase A 1 activity is present. This soluble preparation of lung lysosomal phospholipase A 1 was used to study inhibition by amiodarone and desethylamiodarone, in vitro. Both were extremely potent inhibitors of the lung acid phospholipase A 1. To evaluate the levels of amiodarone in lung lysosomes, rats were treated with the agent for 3 days and the combined mitochondrial/ lysosomal fraction of lung tissue was prepared by differential centrifugation. This fraction had been shown previously to be highly enriched in amiodarone. Purified mitochondria and lysosomes were isolated from the combined mitochondrial/lysosomal fraction with Percoll gradients and analyzed for their drug content by HPLC. Amiodarone and desethylamiodarone were present in roughly equal amounts, relative to protein, in mitochondria and lysosomes, respectively. Amiodarone appears to differ from other cationic amphiphilic drugs which cause lipidosis because the latter are more highly lysosomotropic. Although amiodarone does not appear to be highly lysosomotropic in lung, it causes lysosomal phospholipid storage because of its ability to concentrate in lung and because it inhibits lysosomal phospholipase A to a much greater extent than other cationic amphiphiles such as diethylaminoethoxyhexestrol, chloroquine and chlorphentermine.

Purification of lysosomal phospholipase A and demonstration of proteins that inhibit phospholipase A in a lysosomal fraction from rat kidney cortex.

Phospholipase A has been isolated from a crude lysosomal fraction from rat kidney cortex and purified 7600-fold with a recovery of 9.8% of the starting activity. The purified enzyme is a glycoprotein having an isoelectric point of pH 5.4 and an apparent molecular weight of 30,000 by high-pressure liquid chromatography gel permeation. Naturally occurring inhibitors of lysosomal phospholipase A are present in two of the lysosomal-soluble protein fractions obtained in the purification. They inhibit hydrolysis of 1,2-di[1-14C]oleoylphosphatidylcholine by purified phospholipase A1 with IC50 values of 7-11 micrograms. The inhibition is abolished by preincubation with trypsin at 37 degrees C, but preincubation with trypsin at 4 degrees C has no effect, providing evidence that the inhibitors are proteins. The results suggest that the activity of lysosomal phospholipase A may be regulated in part by inhibitory proteins. Lysosomal phospholipase A from rat kidney hydrolyzes the sn-1 acyl group of phosphatidylcholine, does not require divalent cations for full activity, and is not inhibited by ethylenediaminetetraacetic acid. It has an acid pH optimum of 3.6-3.8. Neither p-bromophenacyl bromide, diisopropyl fluorophosphate, nor mercuric ion inhibits phospholipase A1. In contrast to rat liver, which has two major isoenzymes of acid phospholipase A1, kidney cortex has only one isoenzyme of lysosomal phospholipase A1.

ATP activation of protein degradation by extracts of crude and purified lysosomal preparations.

Activation of proteolysis by ATP was studied in lysates of crude and purified lysosomal preparations from liver and kidney at acid pH. In the crude system, from kidney, it was found that ATP activates proteolysis over a concentration range of 0.1-2 mM. Up to 4-fold activation was observed. GTP and CTP also activated proteolysis, but to a lesser extent. Proteolysis was inhibited by vanadate and molybdate. Fractionation of the kidney lysosomes on Percoll gradients produced two fractions containing lysosomal marker enzymes. Most of the acid phosphatase and the acid pyrophosphatase were found in the lighter band, while most of the beta-galactosidase and cathepsin activity was found in a more dense band. Proteolysis by lysates of both fractions was activated by ATP and inhibited by vanadate and molybdate. In the dense band proteolysis was also nearly totally blocked by pepstatin, and was enhanced by an inhibitor of pyrophosphatases, sodium fluoride. ATP also activates proteolysis in crude lysosomes from liver, but upon fractionation of this tissue it was found that all the lysosomal enzyme markers are present in the dense fraction obtained from the Percoll gradient. Again, proteolysis by lysates of the purified fractions was activated by ATP and inhibited by vanadate and molybdate. These data indicate that ATP can activate proteolysis at acid pH in a lysosomal milieu containing enzymes which also catalyze its breakdown. In the kidney there may be two lysosomal compartments which separate the enzymes catalyzing ATP breakdown from the proteolytic enzymes, but this is not essential for ATP activation as shown by the data from the liver and the crude lysosomal fractions.

Lysosomal sequestration of cytosolic enzymes and lysosomal thiol cathepsins

Cytoplasmic proteins are degraded with different half-lives in vivo. Large parts of proteins are believed to be degraded primarily in autophagic vacuoles-lysosomal system. However, the mechanism by which cell proteins are delivered to lysosomes and whether such a process might be selective for certain cell proteins are still unresolved. We examined the mechanism of autophagy with isolated autophagic vacuoles. Administration of leupeptin, a inhibitor of lysosomal thiol proteinases, induced the accumulation of numerous autophagic vacuoles in the liver. Highly purified preparation of autophagic vacuoles was isolated by Percoll density gradient equilibrium fractionation of crude lysosomal fractions. When cytosolic enzyme activities in autophagic vacuoles were measured, tyrosine aminotransferase and tryptophan oxygenase with short half-lives, and lactic dehydrogenase and aspartate aminotransferase with long half-lives were detected at similar ratios of enzymes in autophagic vacuoles/cytosol. During the time that cathepsin B plus L activities in autophagic vacuoles are inhibited by the injection of leupeptin, cytosolic enzymes are being accumulated in autophagic vacuoles suggesting that leupeptin blocks intralysosomal proteolysis, and that cytosolic enzymes are sequestered continuously into autophagosomes. Administration of glucocorticoid, which induces the synthesis of tyrosine aminotransferase, tryptophan oxygenase and cytosolic aspartate aminotransferase, selectively increased the sequestration of these enzymes to proportional degrees. Dietary manipulation and administration of insulin, which inhibit the formation of autophagic vacuoles, suppressed completely the accumulation of autophagic vacuoles in liver by administration of leupeptin. Results indicate that there is no selective uptake of cytosolic enzymes into autophagosome. When distribution of lysosomal cathepsin B and L in liver, which are inhibited strongly by leupeptin, was examined immunohistochemically, cathepsin L is found only in hepatocytes, but cathepsin B is localized in sinusoidal cells rather than in hepatocytes, suggesting that cathepsin L plays a most important role in intralysosomal proteolysis in hepatocytes.

Characterization of the phospholipid requirement of a rat liver beta-glucosidase.

The lipid requirement of membrane-bound rat liver beta-glucosidase was investigated using 4-methylumbelliferyl-beta-D-glucopyranoside as the substrate. The enzyme was solubilized and delipidated by sequential extraction of a crude lysosomal fraction from rat liver lysosomes with sodium cholate and ice-cold butan-1-ol. Neither saturated nor unsaturated phosphatidylcholine activated this enzyme. In contrast, acidic phospholipids like phosphatidylglycerol (PtdGro) and phosphatidylserine (PtdSer) were effective activators. For the PtdGro series, fatty acid composition was important, with the shorter chain or unsaturated fatty acid-containing PtdGro species being the best activators. Heat-stable factor (HSF) from Gaucher spleen by itself (1-2 micrograms) had no effect on enzyme activity. However, the same amount of HSF when combined with 10 micrograms of PtdSer markedly stimulated beta-glucosidase activity. In the presence of HSF, di-9-cis-octadecenoyl-PtdGro (1 microgram) or -PtdSer (5 micrograms) provided maximum protection of beta-glucosidase against heat (60 degrees C) inactivation. In the absence of phospholipids, HSF had no effect on the rate of inactivation of the enzyme by the suicide inhibitor conduritol B epoxide (t0.5, 12 +/- 0.5 min); the maximum rate of inactivation was achieved in the presence of a mixture of PtdGro (2.5-5 micrograms) and HSF (t0.5, 2.8 min). The combination of PtdSer (10 micrograms) and HSF (1.3 micrograms) lowered the Km for 4-methylumbelliferyl-beta-D-glucopyranoside from 24 to 2.7 mM. Inhibition of the enzyme by the glucocerebrosidase substrate analogues N-hexyl-O-glucosylsphingosine and glucosylsphingosine was influenced by the activator substances. The inclusion of PtdSer and HSF in the beta-glucosidase assay medium lowered the Ki of N-hexyl-O-glucosylsphingosine 20-fold. The same combination of activators decreased the I0.5 of the enzyme for glucosylsphingosine from 89.4 to 7.6 microM. A study of log (Vmax./Km) versus pH indicated that the PtdSer-HSF pair creates the active site of beta-glucosidase, making apparent three ionizable groups on the enzyme with pK values in the range 4.5-5.1.

Characterization of Subfractions of Autolysosomes Isolated from Rat Liver after Leupeptin Treatment

Autolysosomes were isolated from leupeptin-treated rat liver (Furuno, K., Ishikawa, T., & Kato, K. (1982) J. Biochem. 91, 1943-1950). They were disrupted by hypotonic treatment and subfractionated by centrifugation in a discontinuous sucrose gradient into three distinct parts: the membranes, the soluble contents, and the insoluble remnants. The content fraction contained the bulk of the activities of lysosomal enzymes with a protein yield of about 36%. The membrane fraction, representing about 5% of protein of the autolysosomes, had a high specific activity of acid phosphatase. In SDS-polyacrylamide gel electrophoretic analysis, the autolysosomal membranes showed protein profiles similar to those of normal lysosomal membranes. The aggregates of partially digested cellular components, including organelles, in the autolysosomes were recovered as granular materials with a very high density (designated as the remnant fraction). This fraction accounted for more than half of protein of the autolysosomes but contained little of the activities of lysosomal enzymes. Lipid analyses revealed that the autolysosomes were poor in lipids because the lipid content of the insoluble remnants was very low. Measurements of the rate of protein degradation in vitro in the crude lysosomal fraction and the isolated autolysosomes from leupeptin-treated rat liver showed that proteolysis was suppressed within the autolysosomes. It was suggested that lipids of sequestered cellular components were preferentially digested within the autolysosomes due to the inhibition of proteolytic activity by leupeptin, and the resulting massive accumulation of proteins was responsible for the enhanced autolysosomal density.

Sequestration of cytoplasmic enzymes in an autophagic vacuole-lysosomal system induced by injection of leupeptin.

Administration of leupeptin to rats induces the accumulation of numerous autophagic vacuoles in the liver. Furuno et al. (Furuno, K., Ishikawa, T., and Kato, K. (1982) J. Biochem. (Tokyo) 91, 1485-1494) have recently devised a method for Percoll density gradient equilibrium fractionation of crude lysosomal fractions to isolate a highly enriched preparation of autophagic vacuoles. This system was used to determine whether cytoplasmic enzymes are normally sequestered into autophagic vacuoles in fed animals. Within 30 min following the administration of leupeptin to fed rats, several cytoplasmic enzymes could be demonstrated in vacuolar fractions heavier than mitochondria and normal lyosomes. The activities of tyrosine aminotransferase and lactic dehydrogenase as well as antigens of fructose-bisphosphate aldolase were detectable in fractions with densities of 1.115 to 1.15 g/ml containing cathepsins and acid phosphatase. The cytoplasmic enzymes in these fractions exhibited latency and were sequestered within membranous organelles. Six hours after the administration of leupeptin, the autophagic vacuoles gradually disappeared from these fractions concurrently with the loss of both cytoplasmic and lysosomal marker enzymes. For 6 h after injection of leupeptin the activities of cathepsin D and acid phosphatase increased in autophagic vacuoles and decreased in the postvacuolar lysosomal fraction. Administration of dexamethasone, which induces the synthesis of tyrosine aminotransferase and cytosolic aspartate aminotransferase, selectively increased the sequestration of these enzymes to proportional degrees. Cycloheximide administered simultaneously with leupeptin rapidly inhibited formation of autophagic vacuoles and the sequestrations of both cytoplasmic and lysosomal enzymes. However, when cycloheximide was administered 1 h after leupeptin, the formation of autophagosomes and the sequestration of cytoplasmic enzymes were inhibited but the vacuolar uptake of acid phosphatase and cathepsin D continued to increase for several hours. When cycloheximide was injected 1 h after leupeptin, losses of lactic dehydrogenase and aldolase proteins were observed in autophagic vacuoles isolated 1 and 2 h later.

Appearance of autolysosomes in rat liver after leupeptin treatment.

The administration to rats of leupeptin produced prominent numbers of enlarged and irregularly shaped autolysosomes in hepatocytes. Percoll density equilibration of crude lysosomal fractions from rat livers showed that most lysosomal enzyme migrated form normal lysosomal density fractions toward higher density fractions within 30 min after leupeptin injection. The denser particles were ultrastructurally identified with the autolysosomes. In analysis by differential centrifugation of the liver homogenates, lysosomal enzymes became sedimentable with particles of greater sedimentation rate within 30 min after leupeptin injection. These changes in physical properties of lysosomes reverted to normal after 24 h, and concomitant disappearance of autolysosomes in hepatocytes was observed by electron microscopy. When the time course of distribution of leupeptin in subcellular fractions was analyzed, particle-bound leupeptin shifted with time to larger particle fractions in parallel with the shift of lysosomal enzyme distribution. Upon injection with leupeptin, cathepsin B activity was inhibited by more than 80% for about 3 h and was gradually restored to a normal level after 24 h. Leupeptin treatment caused marked delay of the degradation of endocytosed FITC-labeled asialofetuin in hepatic lysosomes, and its inhibitory effect lasted for over 9 h after the injection. It was suggested that autophagy is probably a normal process of protein degradation in hepatocytes, and because of a retarded digestion of sequestered materials, autolysosomes persisted for a long period and made up the majority of the lysosome population in the leupeptin-treated cells.

Effects of ATP on lysosomes: inhibition of the loss of latency caused by cooling.

Triton-filled lysosomes from rat liver and a crude lysosomal fraction from livers of rats not injected with Triton have been used to study the ability of ATP to protect lysosomes against the effects of incubation in vitro at 37 degrees C. When incubation was carried out in a sucrose-NH4Cl medium at pH 8.0 or in buffer containing 0.15 M KCl at pH 7.4, ATP protected against the loss of lysosomal enzyme latency that occurred at 37 degrees C and also against the additional loss of latency that occurred when the incubated lysosomes were cooled to 0 degrees C. The inhibition of the effects of cooling was found to result from a decrease in the loss of latency caused specifically by cooling the lysosomes through the temperature range of the membrane phase transition (15-0 degrees C); ATP did not affect the basic rate of loss of latency at 0 degrees C. Protection was minimal when incubation was carried out in 1 mM ethylenediaminetetraacetic acid without added divalent metal ion and was enhanced by the addition of either Mg2+ or Ca2+. Kinetic studies were carried out on the ATP-protective action against the effects of cooling by adding ATP to lysosomes that had been incubated at 37 degrees C and that were then cooled to 0 degrees C at various times after addition of the ATP. Susceptibility to the effects of cooling continued to decrease until the ATP had been present at 37 degrees C for approximately 3 min before cooling. The concentration of ATP required for maximum initial rates of protection was found to be 10(-3) M or possibly lower. The rate of decrease in susceptibility to the effects of cooling was much slower when ATP was added at 22 than at 37 degrees C, suggesting that enzyme action may be involved in the protective mechanism. Possible mechanisms for the ATP-protective action are discussed.

Effects of Triton WR-1339 on lipoprotein lipolytic activity and lipid content of rat liver lysosomes.

The characteristics of lipoprotein lipolytic activity in lysosomes after the administration of Triton WR-1339 were studied, and the observed decrease in the density of the particles is discussed. The light mitochondrial fraction prepared from rat liver according to the method of De Duve et al. (Biochem. J. (1955) 60, 604) was used as a crude lysosomal fraction, in which acid phosphatase and lipoprotein lipase activities were concentrated. The lipoprotein lipolytic activity of lysosomes had a pH optimum of 4.0. The activity was strongly inhibited by Triton WR-1339 in vitro at low concentrations, while the acid lipase activity was almost unaffected, though relatively high concentration of the detergent significantly inhibited the latter activity. When Triton WR-1339 administered to rats (150 mg/100 g body weight), the activities of both the lipoprotein lipase and acid lipase of lysosomes from the treated rats decreased to one-third of those of control rats. Low-density and high-density lysosomes were partially purified from the light mitochondrial fraction from Triton WR-1339-treated and silver colloid-treated rats, respectively, by sucrose density gradient centrifugation. The low-density lysosomes (d = 1.00-1.13) from Triton WR-1339 administered rats had approximately 4 and 3 times higher contents of triglyceride and cholesterol, respectively, than the high-density lysosomes (d greater than 1.30) from silver colloid-treated rats. In view of these results and the fact that the density of Triton WR-1339 is quite high (at least d = 1.20), the decrease in density of hepatic lysosomes upon Triton WR-1339 administration cannot be due simply to incorporation of the detergent, and may rather be a result of incorporation and accumulation of some lipid(s) (possibly as lipoprotein) into lysosomes together with Triton WR-1339.

Heterogeneity of lysosomes originating from rat liver parenchymal cells. Metabolic relationship of subpopulations separated by density-gradient centrifugation.

1. A crude lysosomal fraction obtained by differential centrifugation of a rat liver homogenate was subjected to zonal centrifugation in iso-osmotic self-generating gradients composed of modified colloidal silica (Percoll). Analysis of relevant marker-enzyme activities shows a continuous band of considerably purified lysosomal particles in the density range 1.04--1.11 g/ml. 2. A relationship between age and buoyant density of the parenchymal lysosomal subpopulations is indicated by the distribution of 125I-labelled asialoglycoproteins in the heterogeneous lysosomes during the catabolism of the glycoprotein. The labelled asialoglycoprotein first appeared in lysosomal particles of low density, which with time progressively acquired a higher density. Furthermore, 30 min after administration the 125I-labelled asialocaeruloplasmin recovered in the light lysosomes was less degraded than the material recovered in the heavy lysosomes. 3. A lysosomal enzyme (arylsulphatase) was found to possess considerably higher isoelectric points in the heavy lysosomes than in the light lysosomes, which is consistent with a relationship between age and density of the lysosomes.

Subcellular localization of the heat-stable glucocerebrosidase activator substance in gaucher spleen

The spleen in Gaucher's disease contains relatively large quantities of a heat-stable activator of the glucocerebrosidase of normal human tissues (Ho, M. W., and O'Brien, J. S. (1971) Proc. Nat. Acad. Sci. USA 68, 2810–2813) that has been shown to be an 11,000 molecular weight acidic glycoprotein (Peters, S. P., et al. (1977) J. Biol. Chem. 252, 563–573). In an effort to determine the subcellular location of the activator, a mannitol-sucrose homogenate of fresh, unfrozen spleen obtained from a 26-year-old patient with adult, nonneuropathic (Type 1) form of Gaucher's disease was subjected to subcellular fractionation. The tissue used in these experiments exhibited a β-glucocerebrosidase deficiency (11% of control tissue characteristic of Gaucher's disease. Mitochondrial and lysosomal fractions obtained by centrifugation of the spleen homogenate at 6900 and and 20,000 g, respectively, contained greater than 80% of the recovered acid phosphatase and heat-stable glucocerebrosidase activator activities. In addition, 60% of the residual glucocerebrosidase activity was recovered in the mitochondrial and lysosomal fractions. The lysosomal and mitochondrial fractions were subjected to equilibrium sucrose density gradient centrifugation. Analysis of the sucrose gradient of the crude mitochondrial fraction demonstrated the mitochondrial marker enzyme (cytochrome oxidase) banding with a specific gravity of 1.19 g/ml, whereas the heat-stable activating factor banded in an acid phosphatase-rich fraction having a specific gravity of 1.12 g/ml. Sucrose gradient analysis of the crude lysosomal fraction obtained from differential centrifugation indicated the activating factor banding with a specific gravity of 1.12 g/ml. Coincident with the activating factor was glucocerebrosidase and acid phosphatase activity. Electron microscopic examination of fractions from each of the sucrose density gradients demonstrated that the glucocerebrosidase activating factor was located in the same acid phosphatase-rich fractions that contained the characteristic Gaucher deposits. Furthermore, when Gaucher deposits were isolated and purified independently by a sucrose gradient procedure, they were found to contain high concentrations of the heat-stable glucocerebrosidase activator. The specific activity of the glucocerebrosidase activating factor was approximately 15-fold greater in the extensively purified Gaucher deposits than in the crude extract of Gaucher spleen from which the deposits were isolated. These observations indicate that the heat-stable activator is associated with the storage deposits contained in lysosomes of the Gaucher cell.

Arylsulphatase activity of corn (Zea mays L.) seedling roots

The crude lysosomal fraction of corn seedling root tips contains an arylsulphatase (E.C. 3.1.6.1) which hydrolysed p-nitrophenyl sulphate at pH 8.0 but had no activity towards p-nitrocatechol sulphate. The Km value for p-nitrophenyl sulphate was 1.24 mM. The hydrolysis of p-nitrophenyl sulphate was linear up to 2 h and the rate was proportional to the amount of enzyme added. The enzyme was strongly inhibited by cyanide, fluoride and phosphate ions and did not resemble the arylsulphatases of bacterial and animal origin.

Ecto-ganglioside-sialidase activity of herpes simplex virus-transformed hamster embryo fibroblasts.

Cellular location of ganglioside-sialidase activity was determined in confluent hamster embryo fibroblasts transformed with herpes simplex virus type 2. Approximately equal specific activities of ganglioside-sialidase activity were found to be associated with the crude lysosomal and crude plasma membrane fractions isolated from whole cell homogenates. Whole transformed cells hydrolyzed exogenous ganglioside substrate, suggesting a partial location of the cellular sialidase on the outer surface of the plasma membrane of these cells. Intact cells were treated with the diazonium salt of sulfanilic acid, a nonpenetrating reagent inhibitory to ecto-enzymes (DePierre, J.W., and M. L. Karnovsky. 1974. J. Biol. Chem. 249:7111-7120). Cytoplasmic lactate dehydrogenase activity was not inhibited by this treatment, and mitochondrial succinate dehydrogenase activity was inhibited only 10%, indicating that intracellular enzymes were not affected. 5'-Nucleotidase activity was diminished 90%, and sialidase very rapidly lost 40% of its exogenously directed activity. These results show that, in herpes simplex virus-transformed fibroblasts, ganglioside-sialidase is both a lysosomal and a plasma membrane enzyme. The plasma membrane sialidase is capable of acting on endogenous plasma membrane sialolipids and also functions in the cultured transformed cell as an ecto-enzyme which can attack exogenous substrates.

Phospholipase A activity in human and ovine uterine tissues

Phospholipase A was assayed in crude lysosomal fractions of human decidua, amnion, chorion and myometrium. Activity was present in all the tissues and was highest in decidua and amnion which contained enzymes with pH optima of 6.5 – 8.0 and 7.2 respectively. Comparison of the activities in tissues obtained before labor with others obtained during labor showed no differences. In sheep, the fetal membranes contained a greater activity of phospholipase A than placenta and myometrium. Stimulation of the fetal adrenals with corticotrophin caused a marked increase in activity in both amnion and chorioallantois. It is concluded that the human amnion and the ovine amnion and chorioallantois could participate in the biosynthesis of prostaglandins by releasing stored arachidonic acid.

The hyaluronidase of brain.

Abstract— Hyaluronidase (hyaluronate glycanohydrolase, EC 3.2.1.35), with a pH optimum of 3.7, was detected in rat and bovine brain. It degraded hyaluronic acid and, at a slower rate, chondroitin sulphate to a mixture of higher oligosaccharides with N‐acetylhexosamine at the reducing end. The enzyme was enriched 5‐ and 6‐fold in a crude lysosomal fraction of rat brain or bovine cerebral cortex, and was further purified to a total enrichment of 9‐fold by ammonium sulphate fractionation. The enzyme activity in grey matter was more than twice that found in white matter, and there was no significant change in enzyme activity as a function of increasing age from the neonatal to the adult rat brain. The level of hyaluronidase activity in rat brain is considerably greaterthan that required to account for the rate of catabolism of hyaluronic acid and chondroitin sulphate measured in vivo.