Triton-filled Lysosomes Research Articles

Identification and characterization of lysosomal enzymes involved in the proteolysis of phenobarbital-inducible cytochrome P450.

We have studied lysosomal proteases capable of degrading a major form of cytochrome P450 (CYP2B1) which was purified from the liver microsomes of phenobarbital-treated rats. After incubation of CYP2B1 with extracts of triton-filled lysosomes (tritosomes), its proteolysis was measured by a quantitative immunoblot procedure. A CYP2B1 protein band with an apparent molecular mass of 53 kilodaltons (kDa) was degraded to 42-, 38-, and 29-kDa polypeptides after a short period of incubation. These proteolytic fragments disappeared on prolonged incubation. One milligram of tritosomal protein contained enough activity to hydrolyze approximately 67.3 micrograms CYP2B1 protein/min at pH 4.5. Approximately 85% of the hydrolyzing activity was localized in a soluble fraction of the tritosomes. The degradation of CYP2B1 was effectively inhibited by pepstatin A, an aspartic protease inhibitor, but not by phenylmethanesulfonyl fluoride (PMSF), o-phenanthroline and leupeptin. CYP2B1-hydrolyzing activity was coeluted with cathepsin D when the soluble fraction was chromatographed by means of Ultrogel Ac44 gel filtration. Cathepsin D, purified from rat livers, was able to degrade CYP2B1 at pH 4.5 which corresponds to the intralysosomal pH. These results indicate that cathepsin D is responsible for the major CYP2B1-hydrolyzing enzyme activity in lysosomes.

Isolation and characterization of a novel membrane glycoprotein of 85,000 molecular weight from rat liver lysosomes.

We have purified and characterized a novel glycoprotein (r-lamp-3) with an apparent molecular weight (Mr) of 85,000 from membranes of triton-filled lysosomes (tritosomes) by the use of immunoaffinity chromatography on a column of monoclonal antibody-Sepharose 4B. r-lamp-3 accounted for approximately 4% of the total proteins in tritosomal membranes. The isoelectric point (pI) of r-lamp-3 was 4.5 and it was shifted to 6.5 after neuraminidase treatment with its molecular weight decreased by about 7000. Pulse-chase experiments in cultured rat hepatocytes using [35S]methionine showed that r-lamp-3 was initially synthesized as a 77,000 polypeptide and processed to a mature protein with an Mr of 85,000. Upon treatment with endo-beta-N-acetylglucosaminidase H (Endo H), the precursor and mature forms were converted to 55,000 and 73,000 polypeptides, respectively. From the Mr reduction of the precursor form, we estimated the presence of 10--12 N-linked oligosaccharides/r-lamp-3 polypeptide. The data on enzymatic deglycosylation suggested that the mature form of r-lamp-3 contained the same numbers of high mannose-type and complex-type N-linked oligosaccharide chains.

Purification and characterization of a major glycoprotein in rat liver lysosomal membrane.

A major lysosomal membrane glycoprotein (LGP107) which has an apparent molecular weight (Mr) of 107 kilodaltons (kDa) was purified from rat liver by a simple method with a yield of 1 mg/87 g wet weight of liver. The purification procedures include; preparation of tritosomal membranes of triton-filled lysosomes (tritosomes), extraction of tritosomal membranes by Lubrol PX, wheat germ agglutinin (WGA)-Sepharose affinity chromatography, and monoclonal antibody-Sepharose affinity chromatography. The quantitative immunoblot analysis indicated that LGP107 represents 6.2% of the total protein of tritosomal membranes. The isoelectric point of the purified glycoprotein was 2.7, and it moved toward neutral pH after sialidase treatment, with its molecular weight decreased by about 10 kDa. LGP107 contained 52% carbohydrates, and the carbohydrate moiety was compared of Fuc, Man, Gal, GlcNAc and sialic acid in a molar ratio of 7.2:68.2:40.6:63.0:32.3, respectively, indicating that LGP107 was highly glycosylated with N-linked complex-type olgosaccharide chains. Out of the N-linked glycans released from the glycoprotein by hydrazinolysis/N-reacetylation, about 70% was sialylated. Anion exchange and reverse-phase high performance liquid chromatography analysis on the structure of N-glycans revealed that a disialyl biantennary form is a major component in the oligosaccharide chains of LGP107.

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.

Cellular changes associated with triton WR1339 accumulation in rat hepatocytes: III. Cellular clearance

Following accumulation of Triton WR1339 by the lysosomes of rat hepatocytes after a single intraperitoneal injection, normal cellular morphology was reestablished gradually over a period ranging beyond 30 days postinjection. This process involved the reduction in the size and numbers of Triton-filled hepatocyte lysosomes from 15 to 30 days postinjection, parallel observation of debris histochemically positive for acid phosphatase, and aryl sulfatase activity in the bile canaliculi and space of Disse, and the reappearance of pericanalicularly oriented dense body lysosomes. By 60 days postinjection the Tritontreated hepatocytes were morphologically indistinguishable from untreated controls. Morphological observation suggested that exocytosis of Triton-filled lysosomes into the extracellular space surrounding hepatocytes, most notably the bile canaliculi and space of Disse, might represent the mechanism by which normal cellular morphology was reestablished.

Mechanisms of loss of latency of lysosomal enzymes. Effects of incubation on the properties of lysosomal membranes.

1. The effects of sucrose and KCl on the loss of latency of lysosomal enzymes caused by incubation at 37 degrees C, pH 7.4, were examined by using Triton-filled lysosomes from rat liver and two fractions from livers of rats not injected with Triton. 2. After incubation, the percentage free activity of lysosomal enzymes was measured before and after cooling to 0 degrees C in order to determine the amount of latency lost at 37 degrees C without cooling and the additional amount lost on cooling the incubated lysosomes to 0 degrees C. 3. The latency that is lost without cooling is first decreased and then increased by increasing the osmotic strength of the incubation medium with KCl, or with sucrose in the presence of KCl. However, if the osmotic strength is increased with sucrose alone, loss of latency is decreased up to 0.25M-sucrose, but is increased only slightly at higher sucrose concentrations. Apparently the lysosome is permeated by hyperosmolar KCl but not by sucrose during incubation. 4. If the osmotic strength of the assay medium is increased with KCl, the loss of latency caused by incubation for 60 min in hyperosmolar KCl is repressed. Thus it appears that a KCl-permeated lysosome can be obtained which is relatively stable until exposure to lower osmolarities. 5. The loss of latency caused by cooling incubated lysosomes to 0 degrees C is largely eliminated if the osmotic strength of the medium in which the lysosomes are cooled is raised sufficiently with either sucrose or KCl. 6. Osmotic-fragility curves were obtained after incubation for 1 and 60 min at iso-osmoticity (0.2M-KCl or 0.25 M-sucrose). Although little loss of latency occurs at iso-osmoticity, lysosomes incubated for 60 min display greatly increased fragility on exposure to hypo-osmolar KCl, hypo-osmolar sucrose or hyperosmolar KCl. 7. It is suggested that permeability to KCl at 37 degrees C and the increase in fragility on exposure to hypo-osmolar conditions are both consequences of injury, probably from enzymic action, sustained by the lysosomal membrane during incubation at 37 degrees C.

The temperature-dependence of the loss of latency of lysosomal enzymes

1. When Triton-filled lysosomes from rat liver are incubated for up to 50min at 37 degrees C, pH7.4, in 0.25m-sucrose, no loss of latency of N-acetyl-beta-glucosaminidase or p-nitrophenyl phosphatase occurs unless the incubated lysosomes are cooled to approx. 15 degrees C. 2. It is suggested that a phase change takes place in the incubated lysosomal membranes on cooling; it starts at approx. 15 degrees C and probably is not complete at 0 degrees C. 3. Incubation of the lysosomes causes an increased potential for loss of latency of the lysosomal enzymes. This potential is not fully expressed at elevated temperature (e.g. 37 degrees C), but is expressed on cooling. 4. The increase at elevated temperature in potential for loss of latency exhibits biphasic kinetics, with an initial rapid phase followed by a slower phase, which is linear with respect to time. The extra loss of latency resulting from the rapid phase in proportional to the temperature of the incubation. 5. Arrhenius plots of the increase is potential for loss of latency during the slow phase for N-acetyl-beta-glucosaminidase and p-nitrophenyl phosphatase exhibit marked deviations from linearity beginning at approx. 15 degrees C. This suggests that the increase in potential for loss of latency is affected by a phase change that occurs around this temperature. 6. Activation energies for the increase in potential for loss of latency at and above 22 degrees C are 53.1+/-5.4kJ/mol (12.7+/-1.3kcal/mol) for N-acetyl-beta-glucosaminidase and 45.2+/-7.5kJ/mol (10.8+/-1.8kcal/mol) for p-nitrophenyl phosphatase. It is postulated that these energies reflect enzymic action, the products of which cause loss of latency to occur on cooling.

Formation of triton WR 1339-filled rat liver lysosomes: II. Involvement of autophagy and of pre-existing lysosomes

The participation of endocytosis in the formation of Triton-filled lysosomes was followed after injecting Triton WR 1339 simultaneously with colloidal gold or horseradish peroxidase. According to cytomorphometric measurements Triton WR 1339 also significantly enhances autophagy. The analysis of the influence of Triton WR 1339 subfractions shows that autophagy is only augmented by the polymer component (‘macrotriton’) but not by the low molecular component (‘microtriton’) alone. When the latter was injected combined with either macrotriton or colloidal gold particles, autophagy increased to a level observed with Triton WR 1339 and beyond the levels determined for either component alone. Thus, autophagy appears to be stimulated by endocytosis. Autolysosomes are transformed within a few hours to peribiliary ‘dense bodies’. These do not display any significant turnover rate within a period of several days; therefore, dense bodies could be prelabelled with colloidal gold in order to show that they are the target organelles ingesting (macro-)Triton. According to difference spectra autophagy leads to a considerable concentration of microsomal and mitochondria! cytochromes in (macro-)Triton-filled lysosomes far beyond the level detected in ‘normal’ lysosomes. Because of the participation of both hetero- and autophagy in the formation of Triton WR 1339-filled lysosomes they have to be classified as telolysosomes.

Glycosphingolipids in Secondary Lysosomes Prepared from Rat Liver

Glycosphingolipid classes were isolated from unfractionated liver and from the buoyant fraction of secondary lysosomes following pretreatment of rats with Triton WR-1339. They were identified by thin-layer chromatography and by sugar analysis using gas chromatography and were quantified by estimating long chain base. Hematoside containing N-acetyl neuraminic acid, glucosyl, lactosyl, trihexosyl, and galactosamine-containing tetrahexosyl ceramides were identified in unfractionated liver in the following concentrations, respectively: 0.89, 0.59, 0.19, 0.06, and 0.1 nmol/mg protein. The same glycosphingolipid classes were identified in buoyant Triton-filled lysosomes where their respective concentrations in terms of protein were 8.6, 21, 28, 34, and 28 times as great as in unfractionated liver. The lysosome fraction was enriched 28-fold in acid phosphatase. These results demonstrate that some glycosphingolipid classes are concentrated in secondary lysosomes to a degree expected of markers for primary lysosomes and plasma membrane. Secondary lysosomes are thought to be derived from these latter two structures.

Changes in lipid composition of triton-filled lysosomes during lysis: Association with activation of acid-active lipases and phospholipases

1. 1. Triton-filled lysosomes were isolated from livers of rats by isopycnic centrifugation. Incubation of these organdies at 37 δC, pH 5.0, resulted in release of β-glucuronidase and a dramatic elevation of lysophospholipids and free fatty acids. At pH 6.4 only minimal changes of this type were seen. 2. 2. Soluble phospholipase A from hepatic lysosomes is inhibited by albumin, protein of the supernatant, heparin and protamine. 3. 3. Defatted albumin inhibits production of lysophospholipids and free fatty acids and the release of β-glucuronidase. 4. 4. Thus, acid-active lipases of lysosomes are activated by incubation at acid pH and appear to contribute significantly to the process of lysis of these organelles. Inhibition of these acid-active lipases results in a stabilization of the lysosomal membrane.

Specificity and Other Properties of Lysosomal Lipase of Rat Liver

Abstract The pH profile obtained for enzymes in rat liver homogenate which hydrolyze glycerol esters of decanoic acid shows two distinct peaks of activity, one at pH 5 to 5.2 and the other at pH 8.6 to 9. The activity at acid pH is associated with lysosomes, and that at alkaline pH with microsomes. Glycerol tri-, 1,2-di-, and 1-monodecanoate are hydrolyzed by lysosomes in decreasing order of activity, and by microsomes in increasing order of activity. When soluble lysosomal lipase hydrolyzed glycerol tri(oleate-1-14C), the percentage of radioactivity recovered in the products was as follows: oleic acid, 40%; glycerol 1,2-dioleate, 52%; glycerol 1,3-dioleate, 8%; and glycerol monooleate, 0%. Complete solubilization of lysosomal lipase is achieved by suspending the lysosomal pellet in 0.05 m sodium phosphate buffer, pH 6.8. Lysosomal lipase is strongly inhibited by certain sulfhydryl reagents. A comparison of lipase activity in liver lysosomes with that in Triton WR 1339-filled liver lysosomes, as well as the limitations imposed by the use of Triton-filled lysosomes, is discussed.

Triglyceride breakdown in rat liver the demonstration of three different lipases

Triglyceride hydrolysis by homogenate fractions of rat liver was measured by determining free fatty acids and glycerol after incubation with artificial triolein or tributyrin emulsions. Triolein lipolysis exhibited two pH maxima at pH 5 and 8.5, respectively. Acid lipase was shown by differential centrifugation to be of lysosomal origin and was purified 300-fold from Triton-filled lysosomes. This enzyme could be activated by sonication and by hypotonic treatment like other lysosomal enzymes. Alkaline lipase was found to sediment with the microsomes. In this fraction hydrolysis of tributyrin was more than 6 times faster than lipolysis with triolein as substrate. The ratio of free fatty acids to glycerol released was above 3 at acid pH and below 3 at alkaline pH. Heparin was shown to stimulate triolein hydrolysis with a maximum at pH 7.5. This heparin-sensitive lipase could be localized in a plasma membrane preparation and could only be demonstrated in the presence of Ca 2+. The results suggest the existence of three distinct lipases in rat liver with different pH maxima and subcellular localization. Their activities are compared with data reported in the literature, and a possible physiological role of the lipases is discussed.