A comparative biochemical research in the Schistocephalus solidus (Cestoda)--three-spined stickleback Gasterosteus aculeatus L. system

The activity of five lysosomal hydrolases (acid phosphatase, DNAase, RNAase, beta-glucosidase, beta-galactosidase), alkaline phosphatase and aldolase have been examined in tissues of the cestode Schistocephalus solidus (Müller, 1776) and the three-spined stickleback Gasterosteus aculeatus (L.) forming a stable parasite-host system. As a rule, the activity of enzymes was higher in a cestode body than in fish tissues. The acid and alkaline phosphatases were the exception. The activity and variation of lysosomal nucleases and aldolase in the parasite differed notably from those in both infested and healthy hosts. The paper discusses the role of lysosomal and cytoplasmic enzymes in a cestode adaptation to parasitism, as well as in the mechanisms of the host's chemical and immunological response to infection.     

Biological Time-Related Changes in Tolerance of Male Mice to Hypoxia—II. Orcadian Rhythm of Lysosomal Susceptibility to Hypoxia

Circadian variations of mouse liver, brain and heart lysosomal susceptibility to hypoxia were investigated. Lysosomal disruption during hypoxia was estimated on the basis of the following measurements: changes in percentage free activity of β-galactosidase and acid phosphatase, tissue loss of both lysosomal enzymes and accumulation of serum β-galactosidase. When exposure to hypoxia took place at the end of the rest phase or at the beginning of the active phase, it was accompanied by maximum increase of percent free activity. This, presumably represents a diffusion of enzymes from lysosomes due to altered membrane permeability. However, hypoxia when occurring during the second part of the active phase and first part of the rest phase resulted in tissues loss of lysosomal enzymes and accumulation of serum lysosomal enzymes. This is believed to represent the release of lysosomal enzymes in bulk from damaged or ruptured lysosomal membranes.     

Biological Time-Related Changes in Tolerance of Male Mice to Hypoxia—II. Orcadian Rhythm of Lysosomal Susceptibility to Hypoxia

Circadian variations of mouse liver, brain and heart lysosomal susceptibility to hypoxia were investigated. Lysosomal disruption during hypoxia was estimated on the basis of the following measurements: changes in percentage free activity of β-galactosidase and acid phosphatase, tissue loss of both lysosomal enzymes and accumulation of serum β-galactosidase. When exposure to hypoxia took place at the end of the rest phase or at the beginning of the active phase, it was accompanied by maximum increase of percent free activity. This, presumably represents a diffusion of enzymes from lysosomes due to altered membrane permeability. However, hypoxia when occurring during the second part of the active phase and first part of the rest phase resulted in tissues loss of lysosomal enzymes and accumulation of serum lysosomal enzymes. This is believed to represent the release of lysosomal enzymes in bulk from damaged or ruptured lysosomal membranes.     

Immunocytochemical localization of lysosomal acid phosphatase in normal and "I-cell" fibroblasts

This study represents the first example of immunological localization of lysosomal acid phosphatase. The intracellular localization of lysosomal acid phosphatase was investigated with immunocytochemical methods at the light and electron microscopical level in cultured fibroblasts obtained from normal subjects and from a patient with I-cell disease. Double-labeling studies using fluorescence microscopy showed that acid phosphatase is present in the same organelles as other hydrolases. At the electron microscopic level in control fibroblasts acid phosphatase was found in the rough endoplasmic reticulum, lysosomes, at the plasma membrane, in vesicles just below the plasma membrane and in multivesicular bodies. This localization was comparable with that of other lysosomal enzymes tested (acid alpha-glucosidase, N-acetyl-beta-hexosaminidase, beta-galactosidase). Acid phosphatase labeling was mainly found in association with the lysosomal membrane and with membranous material present within the lysosome. In I-cell fibroblasts the label was present in the same subcellular organelles but always associated with membranous structures. We suggest that the association of acid phosphatase with membranes might explain the normal enzyme activity found in I-cell fibroblasts.     

Effect of adrenaline on kidney lysosome function in rats during prolonged soft tissue crushing

The total and unsedimentable activity of acid DNase, RNase, phosphatase and arylsulfatases A and B was examined in the rat kidneys during long-term compression of soft tissues in the presence of high excitability of the sympathoadrenal system. Injection of adrenalin to rats with trauma reduced the total activity of DNase, acid phosphatase and arylsulfatases A and B, particularly at the late periods of soft tissue compression, whereas the total activity of acid RNase slightly increased as compared with control. Compression of soft tissues after adrenalin preinjection was accompanied by a substantial rise of unsedimentable activity of the lysosomal enzymes under study in the kidneys. The activity of the enzymes in cytosol progressively ascended as the time of soft tissue injury increased.     

Tilorone acts as a lysosomotropic agent in fibroblasts

Tilorone, an amphiphilic cationic compound with antiviral activity perturbed the lysosomal system. In cultured fibroblasts tilorone induced storage of sulfated glycosaminoglycans, enhanced secretion of precursor forms of lysosomal enzymes, inhibited intracellular proteolytic maturation of lysosomal enzymes, and inhibited receptor-mediated endocytosis of lysosomal enzymes. In isolated lysosomes tilorone was found to increase pH and to abolish the ATP-dependent acidification. These effects suggest that tilorone acts like a weak base that accumulates in acid compartments of the cells, raises the pH therein and interferes with lysosomal catabolic activity and with receptor-mediated transport of lysosomal enzymes.     

A new mechanism for prolactin processing into 16K PRL by secreted cathepsin D

Cathepsins are lysosomal enzymes that were shown to release the antiangiogenic fragments 16K prolactin (PRL), endostatin, and angiostatin by processing precursors at acidic pH in vitro. However, the physiological relevance of these findings is questionable because the neutral pH of physiological fluids is not compatible with the acidic conditions required for the proteolytic activity of these enzymes. Here we show that cathepsin D secreted from various tissues is able to process PRL into 16K PRL outside the cell. To specifically target extracellular proteolysis, we used tissues from PRL receptor-deficient mice, which are unable to internalize PRL. As assessed by the use of specific inhibitors of proton extruders, we show that the proteolytic activity of cathepsin D requires local acid secretion driven by Na(+)/H(+) exchangers and H(+)/ATPase. Although it is usually assumed that cathepsin-mediated generation of antiangiogenic peptides occurs in the moderately acidic pericellular milieu found in malignant tumors, we propose a new mechanism explaining the extracellular activity of this acidic protease under physiological pH. Our data support the concept that secreted lysosomal enzymes could be involved in the maintenance of angiogenesis dormancy via the generation of active antiangiogenic peptides in nonpathological contexts.     

Metabolic priorities during starvation: enzyme sparing in liver and white muscle of Atlantic cod,Gadus morhua L

Atlantic cod, Gadus morhua, respond to starvation first by mobilising hepatic lipids, then muscle and hepatic glycogen and finally muscle proteins. The dual role of proteins as functional elements and energetic reserves should lead to a temporal hierarchy of mobilisation where the nature of a function dictates its conservation during starvation. We examined (1) whether lysosomal and anti-oxidant enzymes in liver and white muscle are spared during prolonged starvation, (2) whether the responses of these enzymes in muscle vary longitudinally. Hepatic contents of lysosomal proteases decreased with starvation, whereas those of catalase (CAT) increased and lysosomal enzymes of carbohydrate metabolism and glutathione S-transferase (GST) did not change. In white muscle, starvation decreased the specific activity of lysosomal enzymes of carbohydrate degradation and doubled that of cathepsin D (CaD). The activity of anti-oxidant enzymes and acid phosphatase in muscle was unchanged with starvation. In white muscle neither lysosomal enzymes nor anti-oxidant enzymes varied significantly with sampling position. In cod muscle, antioxidant enzymes, CaD and acid phosphatase are spared during a period of starvation that decreases lysosomal enzymes of carbohydrate metabolism and decreases glycolytic enzyme activities. In cod liver, the anti-oxidant enzymes, CAT and GST, were also spared during starvation.     

Lysosomal enzymes in cells separated from rat testis

Fractions enriched with Sertoli cell (S), germ cells (G), and interstitial cells (I) were separated from rat testis after enzymic treatment and double filtration through nylon meshes. The fractions were analysed for protein content and for enzymic activity of 4 acid hydrolases known to be of lysosomal nature in other tissues. Acid phosphatase activity was preferentially recovered in Fraction G, the highest activity of beta-glucuronidase was found in Fraction I while the activity of aryl sulphatase and beta-N-acetyl-D-glucosaminidase was prominent in Fraction S. With the exception of acid phosphatase, the enzymes were mostly recovered in a subcellular fraction of whole testis homogenate separated between 600 and 27 000 g. The results may reflect the peculiar enzyme composition of the lysosomal apparatus of each cell type.     

Acid phosphatase of HeLa cells: properties and regulation of lysosomal activity by serum.

Although the subcellular distribution profile of acid phosphatase in HeLa cells is typical of a lysosomal enzyme, different lysosomal (70–80%) and supernatant forms (20–30%) have been demonstrated by their differences in pH activity curves, substrate specificities, thermal stability, sensitivity to inhibitors, and kinetics. Enzymes of the lysosomal fraction displayed anomalous kinetics in the hydrolysis of p-nitrophenyl phosphate. The major lysosomal acid phosphatase activity appears to be associated with the membrane.The total acid phosphatase activity in the cell is controlled by the concentration of serum in the medium. The specific activity in the homogenates of cells grown in high serum concentration (30%) is about twice that of cells grown in low serum concentration (1%). This doubling of specific activity holds for the lysosomal enzyme (or enzymes), but little change occurs in the supernatant form (or forms). Two other lysosomal enzymes, β-glucuronidase and N-acetyl-β-d-hexosaminidase, do not increase in specific activity. The serum-dependent formation of acid phosphatase is sensitive to cycloheximide, actinomycin D, and cordycepin. Cycloheximide blocks the increase in enzymatic activity immediately, whereas cordycepin and actinomycin D have no effect for at least 8 h. These findings suggest that de novo protein synthesis is involved in the induction of lysosomal acid phosphatase by serum and that the mRNA for this enzyme is relatively stable.     

Protease inhibitors reduce lysosomal acid phospholipase A1 activity in cultured rat hepatocytes

When rat hepatocytes were cultured in the presence of various specific protease inhibitors, lysosomal acid phospholipase A1 activity decreased progressively. Exposure of the cultured cells to 0.1 micrograms/ml of pepstatin, E 64, leupeptin or chymostatin also reduced the catalytic activities of several lysosomal marker enzymes. Irrespective of the protease inhibitor type employed, acid phospholipase A1 activity reacted most sensitively, followed by acid phosphatase, acid beta-N-acetyl-D-hexosaminidase and acid beta-glucuronidase. Of the protease inhibitors studied, pepstatin appeared to be most potent in reducing lysosomal enzyme activities in cultured hepatocytes. These findings suggest that proteolytic processes at as yet unknown, possibly extralysosomal sites play an important role in the turnover rates of lysosomal enzymes.     

Study of lysosomal enzymes in chick embryo fibroblasts infected with microorganisms of family Halprowiaceae (Chlamydiaceae)

It is shown that infection of chick embryo fibroblasts with agents of paratrachoma and meningopneumonia Halprowiaceae (Chlamydiaceae) causes a sharp decrease of the activities of lysosomal enzymes, e.g. acidic alpha-glucosidase, beta-glucuronidase, beta-galactosidase, alpha-mannosidase, acid phosphatase, etc. The activity of cytosol enzymes (neutral alpha-glucosidase, amylo-1,6-glucosidase) does not change, however. A decrease in the activities of lysosomal enzymes in infected fibroblasts occurs some time later after inoculation and is due to a release of lysosomal enzymes from the fibroblasts into the culture medium, without loss of cell integrity. No changes in the activity of lysosomal enzymes in fibroblasts and culture medium is observed in the case of inoculation of cells with a killed agents, as well as after contact of cells with a suspension of normal chick embryo yolk sacs. The release of lysosomal enzymes from halprowiae-infected chick embryo fibroblasts probably occurs by the exocytosis.     

Sphingomyelinases in human, bovine and porcine seminal plasma

The seminal plasma of man, boar and bull was found to have a sphingomyelinase (SMase) activity hydrolysing [N-methyl-14C]sphingomyelin. The human and porcine enzymes had an acid pH optimum and were not influenced by divalent metal ions or chelating agents. They were closely similar with the lysosomal enzyme in many tissues. The bovine seminal plasma SMase was partially purified. The enzyme was a glycoprotein with pH optimum at 6.5, a broad pI 4.2-4.8 and molecular mass of 160 and 60 kDa, respectively, in native and SDS-PAGE. The enzyme was activated by Co greater than Mn greater than Cd greater than Ni and inhibited by chelating agents, Cu, Fe, Pb and Zn. The enzyme was clearly distinct from the acid lysosomal SMase and the previously described neutral Mg2+-dependent and independent activities. It had a wide distribution in the bull reproductive tissues.     

Lysosomes in skeletal muscle following denervation

Summary The in-vivo uptake of exogenously applied horseradish peroxidase and the activities of the lysosomal enzymes acid phosphatase and cathepsin D were studied histochemically and/or biochemically in innervated and 2–14 day-denervated tibialis anterior muscles of the mouse. The biochemically determined uptake of horseradish peroxidase showed a large increase already 4 days after denervation. The activities of the lysosomal enzymes increased in a more gradual fashion, and only cathepsin D showed an increase in activity when expressed as total activity per muscle. Histochemically horseradish peroxidase was found to be localized in muscle fibres in characteristic spindle-shaped segments after denervation. The main increase in the number of such segments per transverse section of the muscle occurred between 3 and 6 days after denervation. In serial sections these segments frequently showed positive staining also for acid phosphatase.It is concluded that exogenously applied horseradish peroxidase is taken up into the lysosomal system, which after denervation becomes organized into characteristic spindle-shaped segments in the muscle fibres. The endocytic activity of muscle fibres increases early after denervation. This is followed by a more gradual increase in activity of lysosomal enzymes and finally by an organization of the lysosomal system into characteristic spindle-shaped segments. The results are compatible with the working hypothesis that increased endocytosis may initiate lysosomal activation in denervated skeletal muscle.     

Distribution of lysosomal enzymes in different types of rat liver cells.

Eight lysosomal enzymes were measured in different types of rat liver cells. Hepatocytes were purified by low speed centrifugation of a cell suspension obtained by treating the perfused liver with collagenase. Nonparenchymal cells (NPC) were purified by centrifugation after treating the initial cell suspension with pronase, which selectively destroys the parenchymal cells (PC). Kupffer cells were found to attach selectively to tissue culture dishes after overnight culture of an NPC suspension. The specific activity of lysosomal enzymes was generally higher in NPC than in hepatocytes, but the different enzymes were concentrated to different degrees in the NPC. Specific activity of acid phosphatase was 1.7 times higher in NPC than in hepatocytes. Specific activity of acid DNAase, on the other hand, was 8 times higher in NPC than in hepatocytes. Other enzymes showed intermediate values. Assuming that 30% of the liver cells are nonparenchymal it may be calculated that from 7% (acid phosphatase) to 25% (acid DNAase) of the hepatic lysosomal enzymes are present in the NPC. The pattern of lysosomal enzymes in cultured Kupffer cells was similar to that of the NPC from which the Kupffer cells were derived. Cathepsin D and β-glucuronidase were, however, elevated in Kupffer cells as compared with NPC. The enzyme pattern in Kupffer cells was almost identical with that of rat peritoneal macrophages.     

Physicochemical characteristics of the glycosaminoglycan-lysosomal enzyme interaction in vitro. A model of control of leucocytic lysosomal activity.

1. The activities of 30 different lysosomal enzymes were determined in vitro in the presence of the sulphated glycosaminoglycans, heparin and chondroitin sulphate, all the enzymes being measured on a density-gradient-purified lysosomal fraction. 2. Each enzyme was studied as a function of the pH of the incubation medium. In general the presence of sulphated glycosaminoglycans induced a strong pH-dependent inhibition of lysosomal enzymes at pH values lower than 5.0, with full activity at higher pH values. However, in the particular case of lysozyme and phospholipase A2 the heparin-induced inhibition was maintained in the pH range 4.0-7.0. 3. For certain enzymes, such as acid beta-glycerophosphatase, alpha-galactosidase, acid lipase, lysozyme and phospholipase A2, the pH-dependent behaviour obtained in the presence of heparin was quite different to that obtained with chondroitin sulphate, suggesting the existence of physicochemical characteristic factors playing a role in the intermolecular interaction for each of the sulphated glycosaminoglycans studied. 4. Except in the particular case of peroxidase activity, in all other lysosomal enzymes measured the glycosaminoglycan-enzyme complex formation was a temperature-and time-independent phenomenon. 5. The effects of the ionic strength and pH on this intermolecular interaction reinforce the concept of an electrostatic reversible interaction between anionic groups of the glycosaminoglycans and cationic groups on the enzyme molecule. 6. As leucocytic primary lysosomes have a very acid intragranular pH and large amounts of chondroitin sulphate, we propose that this glycosaminoglycan might act as molecular regulator of leucocytic activity, by inhibiting lysosomal enzymes when the intragranular pH is below the pI of lysosomal enzymes. This fact, plus the intravacuolar pH changes described during the phagocytic process, might explain the unresponsiveness of lysosomal enzymes against each other existing in primary lysosomes as well as its full activation at pH values occurring in secondary lysosomes during the phagocytic process.     

Lysosomal nature of hormonally induced enzymes in wheat aleurone cells

The subcellular distribution of the enzymes alpha-amylase, protease and ribonuclease in wheat aleurone layers after treatment with gibberellic acid was determined by differential centrifugation. Of the alpha-amylase 56% was precipitable from cell homogenates, indicating that it is a particulate enzyme. Similar results were recorded with protease. Particulate alpha-amylase showed distinct structural latency, and membrane-rupturing mechanical or chemical treatments were required to release the enzyme in an active form; the results were completely analogous to results with lysosomal enzymes found in animal tissues. The identification of the hormonally induced enzymes as lysosomal suggests that the hormonal mechanism may be more closely associated with extracellular enzyme synthesis rather than with nucleic acid metabolism.     

Induction of apoptosis in human replicative senescent fibroblasts

Cellular senescence is an irreversible growth phase characteristic of normal cells. We have found that human senescent fibroblasts can be induced to undergo programmed cell death (apoptosis) by ceramide, TNF-alpha, or okadaic acid. The most profound effects were induced by TNF-alpha and okadaic acid treatment. In the present study, we also evaluated the contribution of lysosomal activation as a possible mechanism underlying the induction of apoptosis. Four lysosomal enzyme activities were measured: beta-galactosidase, alpha-galactosidase A, beta-glucoronidase, and acid phosphatase. Using an in situ assay, we have found that the activity of beta-galactosidase, which is also a biochemical marker of senescence, is induced in young proliferating fibroblasts following exposure to all three apoptotic inducing agents. The other enzymes were not significantly induced in young fibroblasts following exposure to agents that induce apoptosis. During replicative senescence, three of the four lysosomal enzymes tested (beta-galactosidase, alpha-galactosidase A, and beta-glucoronidase) are constitutively expressed at high levels. TNF-alpha was the only agent that induced lysosomal activity in senescent fibroblasts, of which only alpha-galactosidase A activity was induced. Our studies show that senescent fibroblasts can be induced to undergo apoptosis in a signal-dependent manner. However, the lysosomal enzymes examined do not appear to be correlated with apoptotic induction.     

Acid phospholipase A activities in rat hepatocytes

Cultured rat hepatocytes exhibit acid phospholipase A activity. On the basis of product formation from stereospecifically radiolabeled phosphatidylethanolamine substrates, phospholipases A1 and A2 have been identified with optimal activities at pH 4.5. According to subcellular fractionation studies, the acid phospholipases in hepatocytes appear to be located in the lysosomal compartment. Application of specific inhibitors of the biosynthesis, glycosylation, and translocation of lysosomal enzymes in hepatocyte cultures suggests a half-life of approx. 1 day for the acid lysosomal phospholipase A1. About the same value for the half-life was obtained for the lysosomal marker enzymes, acid phosphatase and beta-N-acetyl-D-hexosaminidase.     

Proteolytic enzyme activity in rat hindlimb muscles in fetus and during post-natal development

The enzymatic activity of two lysosomal enzymes, acid phosphatase and cathepsin D, was determined in fetus and during post-natal development of the rat gastrocnemius muscle in comparison to the histological differentiation of this muscle. The specific activity of cathepsin D and acid phosphatase was 7 and 2.5 fold higher in the muscle during development until 20 days after birth, than that of mature muscle, respectively. A trend of gradual decrease in the activity of these enzymes was observed concomitantly with the differentiation and maturation of the muscle from mononucleated cells in the fetus to myotubes formation at day 1 after birth, followed by the formation of "young" and then striated myofibers in 10- and 20-day old neonates, respectively. However, no correlation could be found between the lysosomal enzyme activity and the developmental stages of the muscle until 20 days after birth. It is suggested that the elevated activity of lysosomal acid hydrolases may be associated with late developmental processes from young to mature myofibers in normal skeletal muscle and not only in various pathological conditions.