Lysosomal Acid Protease Research Articles

Lysosomal enzymes in experimental allergic encephalomyelitis: time course and evidence of the source.

The lysosomal enzymes acid proteinase and beta-glucuronidase, were assayed in spinal cords of rats during the course of experimental allergic encephalomyelitis (EAE). Histological and histochemical examination was carried out versus controls, in selected areas of the same cords biochemically assayed, to look at the distribution of the lysosomal enzyme acid phosphatase. The biochemical assay showed a significant increase of the enzyme activities during the disease and the increase was significantly correlated with the intensity of the disease. The distribution in the nervous tissue of the increase in acid phosphatase activity observed in animals with EAE, suggests that endogenous nervous cells may contribute to the lysosomal enzyme increase in EAE.

Cloning and expression of the gene for the major excreted protein of transformed mouse fibroblasts. A secreted lysosomal protease regulated by transformation.

The major excreted protein (MEP) of mouse fibroblast cells is the 39,000 Mr precursor to a lysosomal acid protease (cathepsin L) induced by malignant transformation, growth factors, and tumor promoters. We have cloned and characterized the gene for MEP from NIH-3T3 cells. This cosmid clone (pcosMMEP), containing the unique 12,000-base pair mouse MEP gene, has been transfected into monkey kidney (CV-1) cells and human epidermoid carcinoma (A431) cells. The stable A4MEP transfectants produce mouse MEP that is an active cathepsin which is secreted, glycosylated, and processed intracellularly to lower molecular weight forms as in the wild-type NIH-3T3 cells. The CVMEP cells (nontransformed phenotype) produce quantities of mouse MEP similar to that found in NIH-3T3 cells, whereas the A4MEP cells (transformed phenotype) produce greater amounts of MEP similar to the levels seen in Kirsten virus-transformed NIH-3T3 cells. The MEP mRNAs from both mouse cells and stably transfected human cells are the same size and have the same single major site for initiation of transcription, indicating that the cloned mouse MEP promoter is active in transfected cells.

Acid thyroglobulin protease activities in human diseased thyroid glands.

The lysosomal acid protease is thought to be a main enzyme in hydrolysis of thyroglobulin. The protease activity of lysosome-rich fractions of various diseased thyroid glands were assayed by the sensitive and reproducible method developed in our laboratory. They included 78 diseased thyroid glands; 37 thyrotoxic goitres, five irradiated thyroids, five Hashimoto's thyroiditis, one granulomatous thyroiditis, six multinodular goitres, 14 'cold' adenomas, four 'hot' adenomas with three paranodular tissues, and six carcinomas. Fifteen paranodular tissues of cold and solitary thyroid nodules were served as controls. The protease activities were significantly increased in thyroid tissues of thyrotoxic goitres, Hashimoto's thyroiditis, adenomas and adenocarcinomas. In thyrotoxic goitres, the protease activity correlated well with the mean area size of colloid follicles. The 'hot' adenomas had higher protease activity than 'cold' ones. The protease activity of multinodular goitres was nearly normal. Only the irradiated thyroid glands had low protease activities. Therefore the high protease activities in hyperfunctioning goitres seem to reflect hormone secretory activities. On the other hand, cold neoplastic goitres seem to have lysosomal protease activities which are not associated with thyroidal secretory process. The assay of acid protease activity will be useful to understand the pathophysiology of lysosomal system in diseased thyroid glands.

The 52-kDa estrogen-induced protein secreted by MCF 7 cells is a lysosomal acidic protease

An estrogen-induced 52-kDa glycoprotein secreted by human breast cancer cells and able to autostimulate the growth of MCF 7 cells has been purified, using monoclonal antibodies, and characterized. The protein contains mannose 6-phosphate signals on its N-linked high-mannose chains, suggesting that it is a lysosomal enzyme. Both the secreted 52-kDa protein and its processed cellular forms (52-, 48- and 34-kDa) were identified as carboxyl proteinases having an optimal activity at pH 3.5 and being specifically inhibited by pepstatin. This protease is characterized by its inducibility by estrogens and its high concentration in proliferative benign and malignant mammary tissue, when detected by immunohistochemistry. The estrogen-induced secretion of this protease may help to understand how estrogens stimulate mammary tumor growth and/or invasion.

Characterization of insulin-degrading activity of intact and subcellular components of human fibroblasts.

We have studied insulin degrading activity (IDA) in cultured human fibroblasts and assessed the effect of various inhibitors of insulin processing on IDA. To evaluate the role of three enzymes of insulin degradation (neutral protease, microsomal glutathione insulin transhydrogenase, and lysosomal acid protease), we subfractionated homogenized fibroblasts into membrane (and nuclei) cytosol, mitochondria, microsomes, and lysosomes. Greater than 90% of IDA was found to be present in the cytosolar fraction containing neutral protease. IDA in intact fibroblasts was completely inhibited by 1 mM N-ethylmaleimide and partially by 0.5 mM dansylcadaverine (75%), 0.5 mM chloroquine (48%), 1 mg/ml bacitracin (32%) and Trasylol (30%). Lidocaine (5 mM) and glucagon (10(-6)M) exhibited about 15% inhibition with minimal inhibition (7%) by nonsuppressible insulin-like activity. Study of similar inhibitors on subfractionated components indicated inhibition of cytosolar enzyme by N-ethylmaleimide (100%), glucagon (30%), chloroquine (41%), nonsuppressible insulin-like activity (30%), Lidocaine (25%), dansylcadaverine (16%), and bacitracin (11%). Incubation of ammonium sulfate-fractionated cytosolar enzyme at 37 C with A14-125I-insulin resulted in generation of two intermediate peaks as early as 1 min. These peaks could be identified by HPLC but not by molecular sieve chromatography. These intermediates exhibited less immunoprecipitability with antiinsulin antibody and receptor binding with liver membrane preparations than intact insulin. Further incubation of A14-125I-insulin with the cytosolar enzyme(s) resulted in reduction of these peaks as well as insulin and formation of 125Iodotyrosine peak. We conclude that human fibroblast is capable of metabolizing cell-associated A14-125I-insulin in a time- and temperature-dependent manner. This process is inhibited by various inhibitors of insulin processing. The bulk of IDA consists of soluble neutral protease(s) with properties similar to other more purified neutral insulin protease preparations. This fraction, similar to the intact fibroblast degrades insulin to two intermediates with similar molecular weight to that of intact insulin but with more hydrophilicity and less binding affinity to antiinsulin antibody and liver membrane than intact insulin.

O4E0948) - Inhibitory effect of sodium salicylate on lysosomal acid proteinase cathepsin E from rat spleen.

O4E0948) - Inhibitory effect of sodium salicylate on lysosomal acid proteinase cathepsin E from rat spleen.

Increased activities of liver cathepsins T and D in carbon tetrachloride-treated rats.

Changes in activities of a new proteinase cathepsin T as well as some other lysosomal acid proteinases and hydrolases were examined in liver homogenate from rats treated with a single hepatotoxic dose of carbon tetrachloride. The most striking changes were several-fold increases of liver cathepsin T and D activities over their levels in untreated rats 3 days after administration of the agent to rats. Increase of cathepsin T was greater than that of cathepsin D at all doses of the hepatotoxin examined. The activities of N alpha-benzoyl-DL-arginine 2-naphthylamide hydrolase, acid phosphatase, beta-galactosidase and beta-glucuronidase in poisoned rat liver were unchanged or only slightly increased. Cathepsin T and D activities were less enhanced in mitochondrial lysosomal fractions than in the homogenate, and were greatly elevated in the supernatant fractions of liver from the treated rats. As judged from the molecular weights, the elevated activities of cathepsins T and D in the treated rat liver could be attributable to the two cathepsins themselves and not to other proteinases. Administration to rats of other hepatotoxic agents, thioacetamide and dimethylnitrosamine, also induced the elevation of the two cathepsin activities in liver, but on partial hepatectomy the activities of liver cathepsins T and D did not show such marked increases. Nonparenchymal liver cell fractions were responsible for almost all the increased activities of liver cathepsins T and D. It is possible that cathepsins T and D play a role in the heterolytic breakdown of hepatocyte molecules following CCl4 poisoning.

Influence of suspension hypokinesia on rat soleus muscle.

Hindlimb hypokinesia was induced in rats by the Morey method to characterize the response of the soleus muscle. Rats suspended for 1-4 wk exhibited continuous and significant declines in soleus mass, function, and contractile duration. Soleus speeding was in part explained by an alteration in fiber type. The normal incidence of 70-90% type I fibers in the soleus muscle was reduced after 4 wk of suspension to 50% or less in 9 of 11 rats. A significant decline in type I myosin isozyme content occurred without a change in that of type II. Other observed histochemical changes were characteristic of denervation. Consistent with soleus atrophy, there was a significant increase in lysosomal (acid) protease activity. One week of recovery after a 2-wk suspension was characterized by a return to values not significantly different from control for muscle wet weights, peak contraction force, one-half relaxation time, and type I myosin. Persistent differences from control were observed in maximal rate of tension development, contraction time, and denervation-like changes.

Characterization of a lysosomal proteinase purified from haploid cells of Physarum flavicomum undergoing encystment

Haploid cells of Physarum flavicomum differentiating to dormant microcysts exhibited prominent Golgi bodies, lysosomes, and autophagic vacuoles digesting intracellular materials. Total intracellular acid proteinase activities, as well as enzyme specific activity, increased during the encystment process. Lysosomes, isolated by fractionation of cell extracts on sucrose density gradients, were identified by their ultrastructural characteristics as well as their content of the following enzymes of high specific activity: acid proteinase, acid β-galactosidase, acid phosphatase, and β-glucuronidase. The lysosomal acid proteinase was chromatographically purified, and its molecular weight was estimated to be 32 000. The proteinase was most stable in the pH range of 2–3 which similarly corresponds to its pH optimum using hemoglobin and Azocasein, respectively, as substrates. Its isoelectric point was about 2. The enzyme exhibited little activity and was unstable at pH 7 and above. The rate of activity of the proteinase was maximal at 55 °C, and good stability of the enzyme was noted up to 45 °C. The proteinase required a thiol reagent for stability. Pepstatin, which specifically affects acid proteinases, inhibited the enzyme. Also, compounds reactive with enzyme thiol groups were highly effective inhibitors of the proteinase. The lysosomal enzyme is an acid (carboxyl) proteinase with essential thiol groups.

Localization and possible activation mechanisms of transglutaminase in the skin.

ABSTRACTThe activity of transglutaminase per unit of wet weight, dry weight, soluble protein, and DNA was determined in three fractions (stratum corneum, malpighii, and dermis) of cow snout skin. The stratum corneum showed the highest enzyme activity among these three fractions.The activity of transglutaminase in the stratum malpighii was ehhanced remarkably by a 36 hour preincubation period at pH 4.5, but the activity in the stratum corneum did not show any increase in enzyme activity under acidic conditions. This ehhancement of enzyme activity was inhibited by pepstatin (100 μg/ml) and chymostatin (100 μg/ml).Relatively high activities of lysosomal enzymes such as β‐glucuronidase, cathepsin B1, and cathepsin D were detected in the stratum malpighii fraction, but these enzyme activities were low in the stratum corneum fraction.To further clarify the nature and activation systems of transglutaminase from stratum corneum and malpighii, transglutaminase solutions with the same activity were prepared from the two layers. Then the same amount of cathepsin D in the crude malpighian transglutaminase solution was added in the crude transglutaminase solution of stratum corneum. Following incubation, the transglutaminase activity of stratum corneum became elevated to the same level of enzyme activity as that of the stratum malpighii. These results suggest the importance of cathepsin D in the activation system of transglutaminase and possible activation machanisms of epidermal transglutaminase in vivo. Transglutaminase in the stratum malpighii may exist in a nonactivated form. During the autolytic stages in the stratum granulosum, a small amount of this nonactivated transglutaminase may be activated by released lysosomal acid proteinases such as cathepsin B1 and cathepsin D. Thereafter, activated transglutaminase could catalyze the formations of ε‐(γ‐glutamyl) lysine cross‐linking in the proteins synthesized in the cytosol of keratinizing cells with ultimate transformation to the membrane‐lining protein, the so‐called marginal band.In addition, most of the transglutaminase in the stratum corneum may still remain in a nonactivated form, since cathepsin D like enzymes may possibly be inactivated or degraded in the stratum corneum in vivo.

Mechanism of Regulation of Human Epidermal Transglutaminase

The activity of crude human epidermal transglutaminase was enhanced remarkably following 24 hr preincubation at low pH (pH 4.5), whereas the pure human epidermal transglutaminase did not show enhancement of enzyme activity at low pHs. Preincubation of pure transglutaminase with rat liver lysosomal fractions (100 microgram/ml) caused a time-dependent enhancement of activity at pH 4.5, up to 4.5 times of the initial activity. This enhancement was specific for lysosomal fractions among the several rat liver subcellular fractions tested. The activity of purified transglutaminase stimulated by lysosomal fractions was inhibited by pepstatin (50 microgram/ml), chymostatin (50 microgram/ml) and EDTA (1 mM). Preincubation of purified transglutaminase with 5 to 100 microgram/ml cathepsin D caused a time-dependent enhancement of activity up to 9.5-fold over control. This enhancement was specific for cathepsin D among the several lysosomal enzymes tested. These in vitro observations suggest possible activation mechanisms of epidermal transglutaminase in vivo. Epidermal transglutaminase may be activated by lysosomal acid proteinases, such as cathepsin B1 and cathepsin D, which are released and activated during the autolytic stages in granular layer in epidermis.

Neurochemical effects of short-term inhalation exposure to vinyltoluene vapor.

Male rats were exposed to vinyltoluene vapor after pretreatment with polychlorinated biphenyl (PCB). Brain and body solvent burdens were in a linear relationship to the exposure level although it changed between the two weeks while the solvent accumulated in the perirenal fat. The pretreatment caused a significantly smaller burden in the fat samples. Lysosomal acid proteinase was above the control range in the brain homogenate in the highest exposure, while glutathione peroxidase and 2',3'-cyclic nucleotide 3'-phosphohydrolase showed a dose-dependent decrease in the homogenate during the first week. Acid proteinase activity in the glial cells increased above the control range only in the PCB-pretreated rats in the first week. Azoreductase increased in the glial cells above the control range only in the first week, and the pretreatment augmented the increase very significantly. All biochemical effects were largely abolished within two weeks of solvent-free period with the exception of an increase in the cerebral RNA at the highest dose level. Vinyltoluene can cause more pronounced neurochemical effects compared to styrene, xylene, or toluene at similar exposure levels.

Circulating lysosomal enzymes and acute hepatic necrosis.

The activities of the lysosomal enzymes acid and neutral protease, N-acetylglucosaminidase, and acid phosphatase were measured in the serum of patients with fulminant hepatic failure. Acid protease (cathepsin D) activity was increased about tenfold in patients who died and nearly fourfold in those who survived fulminant hepatic failure after paracetamol overdose, whereas activities were increased equally in patients with fulminant hepatic failure due to viral hepatitis whether or not they survived. A correlation was found between serum acid protease activity and prothrombin time, and the increase in cathepsin D activity was sustained over several days compared with aspartate aminotransferase, which showed a sharp early peak and then a fall. Circulating lysosomal proteases can damage other organs, and measurement of their activity may therefore be of added value in assessing prognosis in this condition.

Intercellular Messengers in Joint Tissues in Rheumatoid Arthritis

Although the cause of rheumatoid arthritis is uncertain, the mechanisms by which destruction of joint tissues may occur have been studied extensively. The inflammatory responses in rheumatoid arthritis are probably mediated by a variety of different agents which include prostaglandins, leukotrienes, kinins and other peptide mediators, complement components, and immune complexes. The ultimate destruction of proteoglycans and collagen within cartilage similarly depend upon the release of the appropriate degradative enzymes. At one time these were thought to be predominantly lysosomal acid proteinases but emphasis has recently shifted to neutral metallo-proteinases which include specific enzymes capable of degrading collagen or proteoglycans at neutral pH. Under normal conditions these proteinases are in latent form due in part to the presence of a tissue inhibitor of metallo-proteinases (TIMP). During studies of human joint tissues in culture, it has become apparent that products of one cell type may influence the behaviour of other cells. Thus, monocytes and macrophages may produce mediators, such as interleukins, one of which has been called mononuclear cell factor (MCF), which when added to cultures of human articular chondrocytes or synovial cells, markedly enhances production of prostaglandins and metallo-protein-ases while depressing the amount of TIMP. Cultured human synovial tissue produces factors with similar properties, which may in turn be related to mediators such as catabolin, which can be produced by synovium and other connective tissues and which stimulate chondrocytes to degrade their own matrix.The production of these mediators may not only be relevant to rheumatoid arthritis but also to other diseases. Thus, MCF is capable of stimulating prostaglandin production by gingival cells and cells derived from human bone. Moreover MCF is itself capable of inducing bone resorption.Since both normal and diseased tissues are capable of producing and responding to these mediators, these potential degradative interactions must be kept in check in vivo. Glucocorticosteroids may play a role in the natural suppression of these mechanisms, since in vitro they are capable of inhibiting the production of factors as well as their effects on target tissues.Since these factors probably have anabolic activity as well, they may be involved in connective tissue repair after injury. Such intercellular mediators may play important roles in the control of connective tissue turnover, not only in disease states but also in the normal processes of growth and differentiation.

Biochemical effects of carbon monoxide poisoning in rat brain with special reference to blood carboxyhemoglobin and cerebral cytochrome oxidase activity

Male Wistar rats exposed to 1000 ppm carbon monoxide for 3 h showed a rapid removal of carbon monoxide from the blood, and a cerebral cytochrome oxidase activity within the control range immediately after the end of the exposure. The cytochrome oxidase activity decreased while carboxyhemoglobin concentration diminished during the reoxygenation period. The effect might have been caused through a loss of mitochondria by increased lipid peroxidation as cerebral glutathione concentration decreased and lysosomal acid proteinase activity increased in glial cell fractions. The present results seem to indicate that the cerebral cytochrome oxidase may not be specifically inhibited in non-lethal carbon monoxide poisoning despite its proven interactions in vitro.

Proteinase inhibitors suppress the development of experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis (EAE) is an autoimmune inflammatory disease of the central nervous system (CNS) induced in animals by sensitization with white matter or with myelin basic protein (BP)1,2. EAE is dependent on cell-mediated immune (CMI) reactions requiring T-cell sensitization2–4, and demyelination is observed in the vicinity of perivascular infiltrates of mononuclear cells5. However, the mechanisms by which CMI reactions initiate the clinical and pathological expression of EAE have not been well defined. Numerous mechanisms have been proposed for the degradation of myelin, including activated components of the complement system6,7 and the action of enzymes released by inflammatory cells8,9. Among these enzymes, lipases, lysosomal acid proteinases and lymphocyte proteinases have been stressed8–14. Recent reports from this laboratory have suggested that damage to the myelin sheath may be initiated by the release of neutral proteinases, including plasminogen activator, from macrophages activated during an immune response15. If neutral proteinases and, particularly, plasmin, generated from plasminogen by plasminogen activator, have a key role in initiating the clinical signs and pathology of the demyelinating diseases, it might be expected that inhibitors of these enzymes would protect sensitized animals against EAE. Here we have shown that aminomethylcyclohexane carboxylic acid (AMCA), e-amino-caproic acid (EACA) and p-nitrophenylguanidinobenzoate (NPGB), which are inhibitors of plasminogen activator and other neutral proteinases, gave significant protection against the clinical expression of EAE in Lewis rats. Other protease inhibitors gave partial or no protection.

The distribution of lysosomal cathepsin D in cardiac myocytes.

Lysosomal cathepsin D has been localized with the electron microscope employing an indirect immunohistochemical method using peroxidase labeled, monospecific antibody Fab' subunits. The acid proteinase has been demonstrated within secondary lysosomes of cardiac myocytes and interstitial cells, but not in components of the Golgi apparatus or endoplasmic reticulum. Incubations with a variety of peroxidatic inhibitors suggests that the staining that is observed in secondary lysosomes is attributable to the peroxidase-labeled antibody and not to endogenous oxidation of DAB. The protocol outlined here provides a reproducible method to localize the major lysosomal acid proteinase of the heart at the subcellular level.

Inhibition of thyroid cathepsin D activity by pepstatin

The lysosomal acid protease, cathepsin D, is considered to be the primary enzyme within the thyroid which is responsible for the hydrolysis of thyroglobulin and release of active thyroid hormones. Pepstatin has been shown to be a potent inhibitor of cathepsin D in various organs. Therefore, pepstatin may play a therapeutic role in certain disease states. The purpose of our study was to determine the effect of pepstatin on the release of cathepsin D from incubated slices of thyroid tissue. This thyroid tissue was also measured for cyclic AMP concentration. When thyrotropin (TSH) and a phosphodiesterase inhibitor, methylxanthine (MIX), were added to the medium of incubated thyroid tissue, 819 ± 77 U/g cathepsin D was found in the medium compared to 47 ± 7 U/g when no additional agents were added. The addition of 1.43 m M pepstatin to the medium prior to incubation resulted in 100% inhibition of cathepsin D. Pepstatin did not, however, inhibit another thyroid lysosomal enzyme, β-glucuronidase. TSH + MIX-Stimulated thyroid tissue also showed an elevation in tissue cyclic AMP concentrations after incubation as compared to nonstimulated tissue (6.43 ± 0.58 pmol/whole thyroid vs 1.18 ± 0.23 pmol/whole thyroid). When pepstatin was added to the TSH + MIX-stimulated tissue, there was a slight reduction in cyclic AMP concentration (3.6 ± 0.67 pmol/whole thyroid). However, this concentration was still significantly greater than in nonstimulated tissue. The effect of chlorpromazine was also studied. This agent inhibited the increase in cyclic AMP in TSH + MIX-stimulated tissue, but had no appreciable effect on cathepsin D or β-glucuronidase release. This study showed that pepstatin specifically inhibits thyroid cathepsin D during incubation of thyroid tissue. However, pepstatin does not inhibit β-glucuronidase or TSH + MIX-stimulated tissue cyclic AMP elevations. Further studies with pepstatin using an in vivo model are warranted.

The guinea pig neutrophil calcium-dependent lysosomal enzyme secretory process: Inhibition by nonsteroid anti-inflammatory agents

A selective release of lysosomal granule-associated β-glucuronidase and acid protease, but not cytoplasmic lactate dehydrogenase, occurs during contact between zymosan-treated serum and cytochalasin B-treated neutrophils. The calcium-dependent aspect of this secretory process is demonstrated by the absence of lysosomal enzyme discharge from neutrophils incubated with zymosan-treated serum in calcium-free Krebs-Ringer phosphate medium containing 7.5 mM glucose, pH 7.4, at 37°. The capacity of the divalent cation ionophore, A23187, to induce lysosomal enzyme release also implies a requirement of calcium for secretion to occur. Further, zymosan-treated serum and ionophore-induced release of β-glucuronidase was accompanied by a marked association of 45CaCl 2 with neutrophils. Indomethacin, diftalone, naproxen, ketoprofen and suprofen impeded A23187-stimulated extracellular extrusion of lysosomal enzymes from, and 45CaCl 2 association with, neutrophils. Acetylsalicylic acid and flazalone were inactive. The inhibitory effects of these nonsteroid anti-inflammatory agents could be reversed by increasing the extracellular calcium ion concentration. The specificity of calcium in reversing the effects of these agents was indicated by the observation that supplementing the incubation medium with magnesium would not attenuate the action of these nonsteroid agents. Therefore, the regulation of the selective secretion of lysosomal enzymes from neutrophils by nonsteroid anti-inflammatory agents may be related to their capacity to modulate the association of calcium with these cells.

Labilization of cerebral lysosomes by autologous microsomes and protection by cytosolic fraction and a superoxide scavenger in vitro

Incubation of cerebral lysosomes with autologous microsomes resulted in labilization of the lysosomal complex as assayed through determination of lysosomal acid proteinase activity in the incubate supernatant. Addition of cytosolic fraction with significant glutathione peroxidase activity largely prevented the labilization whereas an artificial scavanger of active oxygen (benzoic acid) was not as effective. Frozen and thawed lysosomes served as controls. The labilization of lysosomes by microsomal oxygen activation mechanism in the absence of exogenous substrate may explain neuronal degeneration in cases of induction of the oxidative activity without generation of reactive metabolites from the exogenous substrate.