Harvesting and separation of two populations of lysosomes from porcine endometrium

The lysosomes present in homogenates of porcine endometrium epithelium equilibrate in two density regions of Percoll gradients. Patterns with varying proportions between high and low density peaks are observed, when aliquots of a tissue sample are processed with different all-glass Potter-Elvejhem homogenizers. The described constant-tolerance shearing device (CTSD), in contrast, provides homogenate fractions with higher latencies and steady distribution patterns. They are characteristic for each of the six lysosomal markers and the six other structure-bound enzymes measured in gradient fractions of the particulate matter harvested between 600g and 17,000g. The 17,000g sediments of CTSD homogenates contain more than 40% of the total lysosomal enzymatic activities. Recoveries from Percoll gradients are between 93 and 101%. Enrichments in the high density region range from 35-fold (beta-glucosidase) to 82-fold (acid ribonuclease). Both lysosomal populations exhibit latencies between 89 and 94%. Our results indicate that light lysosomes can be artificially generated by inappropriate homogenization, which should be considered in experiments on the formation and maturation of lysosomes.     

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.     

Effect on lysosomes of invertase endocytosed by rat-liver

The intracellular localization of invertase endocytosed by rat liver was investigated by analytical centrifugation in sucrose and Percoll gradients of mitochondrial fractions originating from rats killed 15 h after injection. After isopycnic centrifugation in a sucrose gradient, invertase is located in higher density zones than acid hydrolases. The difference between the distribution of invertase and that of acid hydrolases increases with the amount of invertase injected. When the invertase dose is sufficiently high, a change of lysosomal enzyme distribution is clearly visible. It consists in the shift of a proportion of these enzymes to higher density regions where invertase is located. The proportion of hydrolase activity affected by invertase is different for each enzyme measured; it is the least pronounced for acid phosphatase, and most for acid deoxyribonuclease and arylsulfatase. A pretreatment of the rat with Triton WR 1339 considerably decreases the equilibrium density of structures bearing invertase. Nevertheless invertase distribution is quite distinct from that of the bulk of lysosomal enzymes that are recovered in lower density zones of the gradient; on the other hand the invertase injection to rats treated with Triton WR 1339 causes a spreading of the acid hydrolase distribution towards higher density zones. The distribution of acid hydrolases and invertase in a Percoll gradient depends on the sucrose concentration of the solvent. It is shifted towards higher densities when the sucrose concentration increases. The phenomenon is more important for invertase. These results are best explained by supposing that invertase accumulates in a distinct population of lysosomes that can be individualized as a result of the density increase they are subjected to by the invertase they accumulate. It is proposed that these lysosomes mainly originate from non-parenchymal cells of the liver.     

125I-Insulin internalization by perfused rat liver: comparison of its subcellular distribution with that of a lysosomally targeted molecule, 125I-asialofetuin

The subcellular distribution of 125I-insulin in the perfused rat liver was compared with the subcellular distribution of the lysosomally targeted asialoglycoprotein, 125I-asialofetuin. The use of Percoll density gradient medium provided excellent separation of lysosomes from the subcellular membrane fractions. Following perfusion with 125I-asialofetuin, a distinct peak of TCA-precipitable radioactivity could be observed in the lysosomal region of the gradient. In contrast, the gradient distribution of TCA-precipitable radioactivity following perfusion with physiological concentrations of 125I-insulin was unimodal, the observed peak corresponding to the distribution of intracellular membrane marker enzymes. Leupeptin, an inhibitor of lysosomal proteolysis, inhibited the degradation of 125I-asialofetuin but had no effect on 125I-insulin degradation. In addition, leupeptin produced a marked increase in TCA-precipitable radioactivity in the lysosome rich region of gradients prepared from livers perfused with 125I-asialofetuin. No such effect was observed following perfusion with 125I-insulin. These findings are consistent with an initial localization of the internalized insulin molecule with the membraneous system of the liver cell rather than the lysosomal system.     

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.     

Isolation of two lysosomal populations from iron-overloaded rat liver with different iron concentration and proteolytic activity

Iron overload results in an accumulation of electron-dense iron-containing particles (IPs) such as ferritin and hemosiderin within the lysosomes of rat liver cells. In order to evaluate the effect or iron overload on lysosomal function, efforts were made to isolate lysosomes with different iron contents by means of ultracentrifugation in Percoll and Metrizamide gradients. Lysosomes isolated on the Percoll gradient were characterized ultrastructurally by a uniform matrix consisting mainly of IPs and these lysosomes contained a high iron concentration and showed a very low proteolytic activity. They may, therefore, constitute, or be equated, with a special type of residual bodies. They were also fragile, as judged by their significant release of enzymes during incubation in vitro. Lysosomes isolated in the Metrizamide gradient contained remnants of sequestered organelles and some IPs. These organelles displayed a somewhat impeded proteolytic activity compared with control lysosomes, as well as preserved membrane stability during incubation in vitro. We suggest that these may be precursors of the heavily iron-laden lysosomes recovered in the Percoll gradient. Our findings demonstrate that different populations of lysosomes exist in iron-overloaded rat liver cells, which show specific characteristics with regard to ultrastructural appearance, iron content and proteolytic activity. Differing iron contents is the most likely reason for their diverging densities and membrane integrities, whereas the difference in proteolytic activity could be a result of varying amounts of degradable substrate.     

Subcellular redistribution of newly synthesized macrophage lysosomal enzymes. Correlation between delivery to the lysosomes and maturation

Cultured mouse peritoneal macrophages were fractionated by two methods at various times after pulse labeling with [35S]methionine. The lysosomal enzymes beta-glucuronidase and beta-galactosidase were isolated from each fraction by immunoprecipitation and electrophoresis on sodium dodecyl sulfate-acrylamide gels. Two distinct peaks of label were obtained on Percoll density gradients. An early appearing peak of low density, containing the precursor forms of both enzymes, co-sedimented with markers for the endoplasmic reticulum, the Golgi apparatus, and the plasma membrane. With time, immunoprecipitable label cosedimented with the bulk of the lysosomal enzyme activity at high density and corresponded to the mature forms of the lysosomal enzymes. By differential centrifugation, newly synthesized enzymes were found predominantly in small particle fractions, unlike the bulk of the lysosomal enzymic activity which was found in larger particle fractions. With increasing time, newly synthesized enzymes were transferred to assume a distribution similar to that of lysosomal enzymic activity. The results suggest that transport of newly synthesized enzymes to lysosomes and conversion to mature forms are closely linked events. Conversion of lysosomal precursors to mature forms occurs either in a prelysosomal vesicle or shortly after reaching the lysosome. The two enzymes follow similar subcellular pathways at similar rates. Also, the macrophage system appears suitable for direct analysis of newly synthesized lysosomal enzymes during subcellular transport.     

Polyamines stimulate lysosomal cystine transport

Lysosomal cystine transport is a carrier-dependent process that, in isolated lysosomes, is stimulated by proton gradients, membrane potential, and millimolar concentrations of divalent cations. The importance of these regulatory factors in vivo is not well established. Polyamines were found to stimulate cystine transport in Percoll gradient purified rat liver lysosomes with spermidine greater than putrescine = cadaverine greater than spermine in order of effectiveness. Maximal stimulation was achieved with 500 microM spermidine. The effects of optimal concentrations of polyamines and divalent cations on cystine transport were not additive. Spermidine stimulated cystine efflux from lysosomes of cultured human diploid fibroblasts, but had no effect on lysosomes of cystinotic fibroblasts which have defective cystine transport. Spermidine did not accumulate within lysosomes in exchange for cystine, had no effect on lysosomal pH, had only slight effects on the lysosomal membrane potential, and had little effect on either methionine or tyrosine efflux. Polyamines are cellular cytoplasmic components that, in physiologic concentrations, stimulate lysosomal cystine transport.     

A simple procedure for the isolation of highly purified lysosomes from normal rat liver

A procedure for the isolation of highly purified lysosomes from normal rat liver is described. The method depends on the swelling of mitochondria when the postnuclear supernatant fraction is incubated with 1 mM Ca2+. The lysosomes can then be separated from the swollen mitochondria by Percoll density gradient centrifugation. The lysosomal fraction obtained by our method was enriched more than 120-fold in terms of the marker enzymes with a yield of 25%. The electron microscopic examination and the measurement of the activities of marker enzymes for various subcellular organelles indicated that our lysosomal preparation was essentially free from contamination by other organelles.     

Intracellular transport and degradation of 125I-tyramine cellobiose-labelled low-density lipoprotein endocytosed in vivo in rat liver cells studied by means of subcellular fractionation

The intracellular transport and degradation of in vivo endocytosed 125I-tyramine cellobiose-labelled low density lipoprotein (125I-TC-LDL) in rat liver cells were studied by means of subcellular fractionation in Nycodenz, sucrose and Percoll density gradients, as well as by means of analytical differential centrifugation. Initially, labelled LDL was located in endocytic vesicles of low densities. Subsequently, acid-soluble and acid-precipitable radioactivities were found in organelles with buoyant densities distinctly lower than that of the main peaks of the lysosomal marker enzymes acid phosphatase and N-acetyl-beta-glucosaminidase. These prelysosomal organelles may represent multivesicular bodies (MVBs). Finally, 6 h after injection and onwards, the acid-soluble radioactivity cosegregated completely with the two lysosomal marker enzymes, suggesting that the degradation products were in secondary lysosomes. The rate of intracellular processing of LDL was very slow compared to that of asialoglycoproteins, suggesting that LDL followed a unique intracellular pathway, that may be specific for this type of ligand.     

Divergent metabolism of human chorionic gonadotropin subunits within rat ovarian lysosomes

Pseudopregnant rats were injected with either native human chorionic gonadotropin or with (125I)-human chorionic gonadotropin and their ovarian homogenates fractionated on Percoll density gradients. The levels of alpha and beta subunits within subcellular fractions were measured using radioimmunoassays specific for each subunit. Radioactivity measurements of fractions obtained from rats injected with (125I)-human chorionic gonadotropin were used as a separate index of alpha subunit distribution. The alpha subunit was primarily restricted to a combined plasma membrane/prelysosomal vesicle fraction. Immunoreactive beta subunit was present at high concentrations within both this plasma membrane/prelysosomal vesicle fraction and within lysosomes. The striking difference in alpha and beta subcellular distribution may arise from differential sensitivities to lysosomal enzymes.     

Biochemical evidence for an endocytically inactive population of lysosomes

The peroxidase dependent, diaminobenzidine (DAB) density shift procedure was applied to the characterization of lysosomes from Chinese hamster ovary (CHO) cells. Peroxidase activity was localized in lysosomes by a 15-18 h internalization period. After treatment with DAB, the distribution of peroxidase activity in Percoll gradients was shifted, as a population, to a higher density. A bimodal distribution which included a low density population was observed for the native lysosomal enzyme beta-hexosaminidase after DAB treatment. A second lysosomal enzyme, alpha-fucosidase, was strongly inhibited by DAB treatment with the residual activity corresponding in distribution to the light beta-hexosaminidase population. The occurrence of a low density lysosomal population after the DAB procedure suggests the existence of an endocytically inactive lysosomal population in fibroblasts. Probable physiological candidates for such a population are discussed.     

Relations between plasma membrane and lysosomal membrane. 2. Quantitative evaluation of plasma membrane marker enzymes in the lysosomes

We have quantified, in cultured rat fibroblasts, the association to the lysosomal membrane of two classical plasma membrane markers, 5'-nucleotidase and alkaline phosphodiesterase I. To isolate highly purified lysosomal preparations, lysosomes were loaded with horseradish peroxidase (2-h cell uptake, 16-h chase) and isolated by isopycnic centrifugation in linear Percoll gradients, followed by a 3,3'-diaminobenzidine-induced density shift in sucrose gradients. Purified lysosomal preparations contained up to 50% of N-acetyl-beta-glucosaminidase of the homogenate. This lysosomal enzyme was enriched 33-fold in the most purified preparations. In the electron microscope, these preparations appeared to be highly purified and only contained organelles filled with diaminobenzidine reaction products. Analysis of purified preparations indicates that 0.5-0.8% of 5'-nucleotidase, but as much as 10.9-14.3% of alkaline phosphodiesterase I activities of the homogenate, are associated with lysosomes. After freezing-thawing, these activities remained essentially membrane-associated. The larger value obtained for alkaline phosphodiesterase I could not be ascribed to other lysosomal enzymes, as no such activity was detected at acidic pH. These two plasma membrane markers are thus unevenly distributed in the lysosomal compartment.     

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 acid hydrolases in subcellular fractions of proliferating vs non-proliferating fibroblasts

We have employed colloidal silica (Percoll) density-gradient subcellular fractionation technique to examine the distribution of lysosomal hydrolases between intermediate vesicles (primary lysosomes) and secondary lysosomes in contact-inhibited non-proliferating vs proliferating chicken embryo fibroblasts. We find that the activities of lysosomal specific enzymes from both phases of growth are distributed within two peaks; however, the relative amounts differ markedly. In normal, non-proliferating cells approx. 60% of the total activities of cathepsin B, beta-mannosidase, alpha-fucosidase, beta-galactosidase and hexosaminidase is recovered in the heavier density fraction corresponding to secondary lysosomes, while less than 9% of the enzyme activities are recovered in the light-density peak. With transformed cells, between 16 and 22% of activity for these enzymes are recovered in the lighter density intermediate vesicle fraction, when less than 40% of the enzyme activities recovered in the heavy density fraction. beta-Glucuronidase distribution was different from that of the above enzymes. First, a more even distribution between the two lysosomal fractions was found with non-proliferating normal cells (33% in heavy-density fraction and 21% in light-density fraction), whereas more than 40% of the total enzyme activity was recovered in the lighter density fraction from transformed cells. Also, the amount of cathepsin B contained in the vesicle fractions is increased severalfold relative to that of contact-inhibited normal cells. However, the apparent differences in enzyme distribution between confluent normal and transformed cells are not found when vesicles are prepared from subconfluent, actively proliferating cultures. We have also compared the Percoll density gradient patterns of membrane vesicles from proliferating and non-proliferating human fibroblasts, since most earlier studies utilized this system. Again, we find that the majority of beta-hexosaminidase activity (41%) of contact-inhibited, confluent cells is recovered in the heavier density fraction with less than 15% in the lighter density fraction. Also, the distribution of beta-hexosaminidase between the heavy density and light density vesicle fractions is altered in homogenates from exponentially growing cells, being 22% and 26% respectively. We conclude that the distribution of lysosomal hydrolases between the two vesicle populations is growth-phase dependent and is markedly heterogeneous in proliferating cells.     

In vitro uptake of particles by lysosomes

Isolated rat liver lysosomes were incubated with Percoll particles in vitro at 25 and 37 °C. On morphological examination the incubated lysosomes contain vesicles some of which enclose Percoll particles, indicating that invagination of the lysosomal membrane occurs in vitro by means of microautophagy. Vesiculation occurs by formation of flaplike processes or cuplike invaginations. At later time points of incubation Percoll particles can be seen free in the lysosomal matrix indicating rupture or digestion of the vesicular membrane. The uptake of isotopically pre-labelled Percoll particles increases with incubation time and temperature.It is concluded that lysosomes show microautophagic activity in vitro and that this may be a mechanism for degradation of soluble cytoplasmic proteins also in vivo.     

Presence of a lysosomal enzyme, arylsulfatase-A, in the prelysosome-endosome compartments of human cultured fibroblasts

Although endosomes and lysosomes are associated with different subcellular functions, we present evidence that a lysosomal enzyme, arylsulfatase-A, is present in prelysosomal vesicles which constitute part of the endosomal compartment. When human cultured fibroblasts were subfractionated with Percoll gradients, arylsulfatase-A activity was enriched in three subcellular fractions: dense lysosomes, light lysosomes, and light membranous vesicles. Pulsing the cells for 1 to 10 min with the fluid-phase endocytic marker, horseradish peroxidase, showed that endosomes enriched with the marker were distributed partly in the light lysosome fraction but mainly in the light membranous fraction. By pulsing the fibroblasts for 10 min with horseradish peroxidase conjugated to colloidal gold and then staining the light membranous and light lysosomal fractions for arylsulfatase-A activity with a specific cytochemical technique, the endocytic marker was detected under the electron microscope in the same vesicles as the lysosomal enzyme. The origin of the lysosomal enzyme in this endosomal compartment was shown not to be acquired through mannose 6-phosphate receptor-mediated endocytosis of enzymes previously secreted from the cell. Together with our recent finding that the light membranous fraction contains prelysosomes distinct from bona fide lysosomes and was highly enriched with newly synthesized arylsulfatase-A molecules, these results demonstrate that prelysosomes also constitute part of the endosomal compartment to which intracellular lysosomal enzymes are targeted.     

Isolation of monkey aortic lysosomes using Percoll density gradient

A novel technique involving the Percoll density gradient and 0.01M phosphate buffer has been employed for the first time on aortic tissue for isolation of lysosomes. The purity of the lysosomes has been established by marker-enzymes, acid phosphatase and N-acetyl-beta-D-glucosaminidase and latent activities of lysosomal hydrolases. The heavier fraction (density 1.08) obtained after Percoll density gradient centrifugation showed high specific activities of lysosomal hydrolases and these enzymes were markedly latent. Moreover this heavier (lysosome rich) fraction has been noted to be free of other sub-cellular contaminants.     

Distribution of chromaffin secretory vesicles, acetylcholinesterase, and lysosomal enzymes in sucrose and Percoll gradients

Crude chromaffin secretory vesicles, obtained by differential centrifugation, were further purified on isotonic (Percoll) gradients. The chromaffin vesicle fractions recovered from the gradients contain acetylcholinesterase as well as lysosomal enzymes. With the aid of a subsequent sucrose gradient lysosomal enzymes could be removed from chromaffin vesicle fractions, but not acetylcholinesterase. This suggests that lysosomal enzymes do not pass through the chromaffin vesicles during the biogenesis of lysosomes but acetylcholinesterase does.     

Identification of two subpopulations of thyroid lysosomes: relation to the thyroglobulin proteolytic pathway.

Using a combination of differential centrifugation and isopycnic centrifugation in Percoll gradients, we obtained a highly purified preparation of thyroid lysosomes [Alquier, Guenin, Munari-Silem, Audebet & Rousset (1985) Biochem. J. 232, 529-537] in which we identified thyroglobulin. From this observation, we postulated that the isolated lysosome population could be composed of primary lysosomes and of secondary lysosomes resulting from the fusion of lysosomes with thyroglobulin-containing vesicles. In the present study, we have tried to characterize these lysosome populations by (a) subfractionation of purified lysosomes using iterative centrifugation on Percoll gradients and (b) by functional studies on cultured thyroid cells. Thyroglobulin analysed by soluble phase radioimmunoassay, Western blotting or immunoprecipitation was used as a marker of secondary lysosomes. The total lysosome population separated from other cell organelles on a first gradient was centrifuged on a second Percoll gradient. Resedimented lysosomes were recovered as a slightly asymmetrical peak under which the distribution patterns of acid hydrolase activities and immunoreactive thyroglobulin did not superimpose. This lysosomal material (L) was separated into two fractions: a light (thyroglobulin-enriched) fraction (L2) and a dense fraction (L1). L1 and L2 subfractions centrifuged on a third series of Percoll gradients were recovered as symmetrical peaks at buoyant densities of 1.12-1.13 and 1.08 g/ml, respectively. In each case, protein and acid hydrolase activities were superimposable. The specific activity of acid phosphatase was slightly lower in L2 than in L1. In contrast, the immunoassayable thyroglobulin content of L2 was about 4-fold higher than that of L1. The overall polypeptide composition of L, L1 and L2 analysed by polyacrylamide-gel electrophoresis was very similar, except for thyroglobulin which was more abundant in L2 than in either L or L1. The functional relationship between L1 and L2 lysosome subpopulations has been studied in cultured thyroid cells reassociated into follicles. Thyroid cells, prelabelled with 125I-iodide to generate 125I-thyroglobulin, were incubated in the absence of in the presence of inhibitors of intralysosomal proteolysis. The fate of 125I-thyroglobulin, and especially its appearance in the lysosomal compartment, was studied by Percoll gradient fractionation and immunoprecipitation. Treatment of prelabelled thyroid cells with chloroquine and leupeptin induced the accumulation of immunoprecipitable 125I-thyroglobulin into a lysosome fraction corresponding to the L2 subpopulation. In control cells, in which intralysosomal proteolysis was n