Lysosomal Acid Hydrolases Research Articles

Purification and characterization of lysosomal alpha-glucosidase secreted by eukaryote Tetrahymena.

A large amount of lysosomal acid hydrolases was released into the medium by Tetrahymena pyriformis strain W during growth. An extracellular lysosomal acid alpha-glucosidase has been purified 500-fold with a 41% yield to homogeneity, as judged by polyacrylamide gel electrophoresis. It was found to be a glycoprotein and to consist of a single 110,000-dalton polypeptide chain. The carbohydrate content of the alpha-glucosidase was equivalent to 2.8% of the total protein content, and the oligosaccharide moiety was composed of mannose and N-acetylglucosamine in a molar ratio of 6.7:2. The optimal pHs for hydrolysis of maltose and p-nitrophenyl-alpha-glucopyranoside, maltose, isomaltose, and glycogen were 1.1 mM, 2.5 mM, 33.0 mM, and 18.5 mg/ml, respectively. This purified enzyme appears to have alpha-1,6-glucosidase as well as alpha-1,4-glucosidase activity. Turanose has a noncompetitive inhibitory effect on the hydrolysis of maltose. The antibody raised against Tetrahymena acid alpha-glucosidase inhibited the hydrolysis of all substrates tested. These properties of Tetrahymena acid alpha-glucosidase were found to be similar to those of the human liver lysosomal alpha-glucosidase.

Peripheral blood mononuclear cells stimulate N-acetyl-beta-glucosaminidase levels of human synovial fibroblast-like cells.

Fibroblast-like synovial cells isolated from intact joints of non-arthritic human donors released up to nine-times higher activity of the lysosomal acid hydrolase N-acetyl-beta-glucosaminidase (NAG) than controls when incubated in conditioned medium from homologous peripheral blood mononuclear cells (MCCM). This increase occurred without decrease in cell numbers or other evidence of cytotoxicity. An increase in cell-associated NAG activity was also suggested, but this was not statistically significant. Indomethacin present during production of MCCM or added with MCCM to fibroblast cultures did not alter the response to MCCM, indicating that the effect of MCCM was not due to the presence of products from the cyclo-oxygenase pathway. At a concentration known to block protein synthesis in most cells (10(-5) M), cycloheximide markedly suppressed the NAG releasing response to MCCM. The secretion of NAG due to MCCM was not affected by all-trans retinoic acid (10(-6) M) but was suppressed by the corticosteroid, dexamethasone.

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.

Phenotypically and functionally distinct subpopulations of human lymphocytes with T cell markers also exhibit different cytochemical patterns of staining for lysosomal enzymes

Human peripheral blood lymphocytes that express T cell markers, when reacted for the cytochemical localization of lysosomal acid hydrolases, display two major patterns of staining, i.e., dot-like and scattered granular. Previous attempts to fractionate T cells according to surface markers have yielded populations of cells with heterogeneous patterns of cytochemical staining. In this study, peripheral blood cells forming rosettes with sheep erythrocytes have been fractionated by sequential staining with two monoclonal antibodies, D12 and 2D2, followed by fluorescence-activated cell sorting. These reagents have been shown previously to recognize a subpopulation of cells capable of suppressing T cell proliferation. All of the cells positive for D12 and 2D2 stained for acid hydrolases with the scattered granular pattern, whereas the large majority of the cells negative for both markers stained with the dot-like pattern. It is concluded that suppressor cells within the E+ cell fraction have the cytochemical characteristics of large granular lymphocytes.

Phenotypically and Functionally Distinct Subpopulations of Human Lymphocytes With T Cell Markers Also Exhibit Different Cytochemical Patterns of Staining for Lysosomal Enzymes

Human peripheral blood lymphocytes that express T cell markers, when reacted for the cytochemical localization of lysosomal acid hydrolases, display two major patterns of staining, i.e., dot-like and scattered granular. Previous attempts to fractionate T cells according to surface markers have yielded populations of cells with heterogeneous patterns of cytochemical staining. In this study, peripheral blood cells forming rosettes with sheep erythrocytes have been fractionated by sequential staining with two monoclonal antibodies, D12 and 2D2, followed by fluorescence-activated cell sorting. These reagents have been shown previously to recognize a subpopulation of cells capable of suppressing T cell proliferation. All of the cells positive for D12 and 2D2 stained for acid hydrolases with the scattered granular pattern, whereas the large majority of the cells negative for both markers stained with the dot-like pattern. It is concluded that suppressor cells within the E+ cell fraction have the cytochemical characteristics of large granular lymphocytes.

Stimulation of glycosaminoglycan production and lysosomal activity of human synovial cells in culture by low environmental pH.

Glycosaminoglycan production, acid hydrolase activity, proliferation, and morphology were examined in human synovial cells subjected to low environmental pH. The amount and the molecular size of newly synthesised glycosaminoglycan (GAG) were increased without significant change in the rate of cell proliferation. Lowered pH produced an increase in the size of cytoplasmic organelles. Some of these possessed ultrastructural features of lysosomes, but others were clearly nonlysosomal and were of uncertain identity. Intracellular activity of the lysosomal acid hydrolase N-acetyl-beta-glucosaminidase (NAG) was not altered by low pH, but a marked increase occurred in extracellular NAG activity, indicating enhanced release.

Increase in the activity of lysosomal acid hydrolases in the chondrocytes of arthritic joints of rabbits with experimental allergic arthritis.

Undecalcified cryostat sections of the cartilage-covered ends of the femurs have been prepared from the control and inflamed knees of rabbits with experimental allergic arthritis. The activities of two lysosomal hydrolases, naphthylamidase and beta-glucuronidase, were assayed in the chondrocytes of the articular cartilage in such sections by scanning and integrating microdensitometry. The activities of both enzymes were found to increase significantly in the chondrocytes of the inflamed joints.

Stimulation of thyroidal thiol endopeptidases by thyrotropin.

Rabbit thyroids contain cathepsin D (CD) and several thiol endopeptidases including cathepsin B and three newly described enzymes (cathepsins 180K, 110K, and 45K). The present paper assesses the relative physiological importance of these enzymes in thyroglobulin degradation in rabbits. Thyroidal thiol endopeptidase [thiol thyroglobulin hydrolase (thiol TgH)] activity increased in the absence of changes in CD activity in animals treated with 10 U bovine TSH. Peak enzyme activity occurred 24 h after injection of hormone. After 20 U bovine TSH, thiol endopeptidase activity increased by approximately 100%, whereas CD increased by 50%. The increase in thiol enzyme activity was attributed both to cathepsin B and to the other thiol endopeptidases. The lysosomal acid hydrolases acid phosphatase and dipeptidyl peptidase II were unaffected by TSH at either dose level. Thiol TgH activity, but not CD activity, was decreased in thyroids of rabbits treated with T4 [5 micrograms/(100 g BW X day)] for 1 week. All thyroidal acid hydrolases examined were suppressed in animals receiving T4 for 3 weeks. Thiol TgH activity was localized primarily to a lysosome-enriched fraction of thyroid homogenates. Our results suggest that the thiol proteases probably are the most important endopeptidases in thyroglobulin hydrolysis in vivo and that their activities are influenced by TSH.

A possible role of protein kinase C in signal-induced lysosomal enzyme release

In platelets, activation of protein kinase C and mobilization of Ca 2+ were selectively induced by the addition of 1-oleoyl-2-acetyl-glycerol and a low concentration of A23187, respectively (Kaibuchi, K., Takai, Y., Sawamura, M., Hoshijima, M., Fujikura, T. and Nishizuka, Y. (1983) J. Biol. Chem. 258 , 6701–6704) . Using this procedure evidence was obtained suggesting that the protein phosphorylation and Ca 2+ mobilization were both essential and synergistically effective to cause release of lysosomal acid hydrolases such as N-acetylglucosaminidase. A similar observation was made for the lysosomal enzyme release from rat neutrophils.

Studies on gentamicin-induced labilization of rat kidney lysosomes in vitro: Possible protection by selenium

The labilizing effect of gentamicin, an aminoglycoside antibiotic, on isolated rat kidney lysosomes was investigated. The light-scattering behavior of lysosomal suspensions and the release of lysosomal acid hydrolase enzymes (acid phosphatase, β-glucuronidase and muramidase) from incubated lysosomal suspensions, in the presence of gentamicin, were used as indices of lysosomal membrane labilization. Gentamicin was found to cause a decrease in light absorbance and a release of lysosomal acid hydrolases, which indicate lysosomal membrane swelling. In the presence of selenium, in the form of potassium selenate, the decrease in light absorbance of lysosomal suspensions and the release of lysosomal acid hydrolases from isolated lysosome particles were reduced markedly. This suggests that selenium protects against gentamicin-induced lysosomal membrane labilization. The possible mechanisms of protection by selenium are discussed.

Hexosaminidase: A marker for intestinal gangrene in necrotizing enterocolitis

Detection of intestinal ischemia, prior to necrosis, is a major clinical problem. The lysosomal acid hydrolase, hexosaminidase (HEX), is known to be elevated in intestinal infarction. To determine if this enzyme could differentiate between partial intestinal ischemia and full-thickness intestinal gangrene, the following rat study was designed. Partial segmental intestinal ischemia was created by ligating alternate vascular bundles over a short (6 vessel) segment of the small-bowel mesentery, and complete segmental intestinal vascular occlusion was achieved by ligating the blood supply to the ileocecal segment. Preoperative serum HEX values were obtained from 15 animals. The rats were separated into one sham-operated and two intestinal ischemia groups. At four hours after surgery HEX values were determined. Total HEX activity was significantly elevated four hours after insult in both partial and complete intestinal ischemia, (P less than 0.005 and P less than 0.001 respectively). Total HEX activity was greater in complete intestinal ischemia than in partial ischemia, (P less than 0.05). Three neonates with intestinal perforation, secondary to necrotizing enterocolitis, were evaluated. The mean preoperative HEX activity was 1421 nmol/hr/mL serum and the mean post-resection HEX activity was 808 nmol/hr/mL serum. These data suggest that serum HEX activity may be a good marker for intestinal gangrene in neonates with necrotizing enterocolitis.

Increased susceptibility to Escherichia coli infection in mice pretreated with Corynebacterium parvum.

The contribution of activated macrophages to protection against Escherichia coli was studied in mice treated intravenously with Corynebacterium parvum 7 days before infection. C. parvum-treated mice showed increased phagocytic activity and enhanced resistance to Listeria infection. In contrast, these mice showed increased susceptibility to a subsequent challenge with E. coli that correlated closely with a reduction in the LD50 of lipopolysaccharide (LPS) in these mice. The peritoneal macrophages obtained from C. parvum-treated mice had a strong ability to phagocytize and kill E. coli in in vitro experiments. A rapid decline in the number of bacteria in the liver of C. parvum-treated mice was observed in the early period of infection. However, the number of bacteria in liver and spleen increased progressively to a lethal dose from 6 hr after infection. At this time, a significant increase in beta-glucuronidase, a lysosomal acid hydrolase, was found in the serum of these mice. In vitro experiments revealed that the peritoneal macrophages from C. parvum-treated mice were highly susceptible to the cytotoxic effect of LPS after 6 hr of incubation with LPS. It is suggested that the hypersensitivity of activated macrophages to the cytotoxic effect of endotoxin derived from E. coli may be partly responsible for the increased susceptibility of C. parvum-treated mice to E. coli infection.

Inhibition of the release of prostaglandins, leukotrienes and lysosomal acid hydrolases from macrophages by selective inhibitors of lecithin biosynthesis

The release of the inflammatory mediators, prostaglandins (PGs), leukotrienes (LT) and lysosomal acid hydrolases (LAH), by macrophages is stimulated by endocytic stimuli such as zymosan. This process can be interfered with by specific inhibitors of phosphatidylcholine (PC) biosynthesis. The diphenylsulfone dapsone and three analogs selectively inhibited [ 14C]choline incorporation into PC but had varied effects on inhibition of mediator release by macrophages. Dapsone inhibited the release of PGs, LT and LAH, whereas the three closely related structural analogs inhibited LT and LAH release only, with little or no effect on PG production.

Electron microscopic cytochemical studies on acid phosphatase activity in acute myocardial ischemia.

Alterations in the localization and the intensity of acid phosphatase activity were studied electron microscopically in acute ischemic myocardial cells in the dog, in relation to processes of cellular degradation. In normal myocardial cells, the acid phosphatase activity was concentrated in the terminal cisternae, the longitudinal elements and the subsarcolemmal cisternae of the sarcoplasmic reticulum and the primary lysosomes. Activity was moderate in the secondary lysosomes, residual bodies and Golgi apparatus. As early as 15 min after coronary ligation the intensity of acid phosphatase activity increased in the enlarged lysosomes and the sarcoplasmic reticulum of the ischemic myocardial cells. Fine deposits of the reaction product were distributed in the sarcoplasm around lysosomes and the sarcoplasmic reticulum after 30 min, and the activity began to decrease in lysosomes and in the sarcoplasmic reticulum. One to 3 hours after ligation, intramitochondrial dense deposits appeared, and the reaction product decreased both in lysosomes and the sarcoplasmic reticulum. The fine reaction product, which leaked from lysosomes and the sarcoplasmic reticulum, was scattered in the sarcoplasm and was accompanied by fine structural changes indicating cellular necrosis. From these findings it is strongly suggested that acid hydrolases in lysosomes and the sarcoplasmic reticulum are closely related to the necrotic process in ischemic myocardial cells.

Intestinal glycosidase activities in one adult and two suckling echidnas: absence of a neutral lactase (beta-D-galactosidase).

The activities of various glycosidases in homogenates of the small-intestinal mucosa of one adult and two suckling echidnas, Tachyglossus aculeatus, were investigated. The activities of lactase (beta-D-galactosidase), beta-N-acetylglucosaminidase, neuraminidase and alpha-L-fucosidase were higher in the sucklings than in the adult animal. Maltase and isomaltase activities were lower. Sucrase and cellobiase activities were absent or present in trace amounts only. The lactase activity had a pH optimum of 4.0-4.5, was predominantly in the soluble fraction following ultracentrifugation and was inhibited by p-chloromercuribenzene sulfonate, suggesting that it was due to a lysosomal acid beta-galactosidase and not a brush-border neutral lactase. The maltase activity of the sucklings also had the characteristics predominantly of a lysosomal acid hydrolase. It is proposed that in suckling echidnas, the oligosaccharides (mainly neuraminyllactose and fucosyllactose) of the mother's milk are digested intracellularly by lysosomal enzymes, rather than at the brush border, of the epithelial cells of the small-intestinal mucosa.

A comparison of lysosomal acid hydrolase activities in human gastric antrum and in different regions of the porcine stomach

1. 1. Lysosomes are known to be implicated in many disease states in both humans and animals. 2. 2. In order to assess the possible role of lysosomes in the aetiology of human gastric ulceration, lysosomal enzyme levels and some of their properties were determined in human gastric antrum and in three discrete porcine gastric regions. 3. 3. Of the four regions investigated, the strongest similarity emerged between the enzymes of the porcine pyloric gland area and those of the human antrum. 4. 4. These data suggest that the pyloric gland is an appropriate model tissue for a comparative in vitro study of lysosomal involvement in human gastric ulceration.

A sensitive fluorometric assay for the simultaneous estimation of pepsin and pepsinogen in gastric mucosa

An assay for pepsin has been developed based on the fluorometric measurement of trichloroacetic acid-soluble peptides released from casein at pH 5.3. The increase in relative fluorescence was most sensitive in the range 10–50 μg pepsin/1 and casein hydrolysis was not affected by the addition of up to a 1000-fold molar excess of pepsinogen. This assay has been used to measure the free and total acid proteinase content of biopsies (< 5 mg) from different areas of the gastric mucosa of rat and man. Interference by the major lysosomal acid hydrolase. cathepsin D, could be eliminated by the differential stability of pepsin and cathepsin D at acid and neutral pH. The free acid proteinase activity of biopsies from the corpus were almost identical in these species whereas the total acid proteinase activity was approximately 5-fold greater in man.

Genetic heterogeneity of I-cell disease is demonstrated by complementation of lysosomal enzyme processing mutants.

I-cell disease (mucolipidosis II) is a fatal childhood disorder affecting the expression of multiple lysosomal acid hydrolases. The disorder is characterized by clinical and biochemical heterogeneity which may reflect different mutants with a similar phenotype. Genetic complementation studies demonstrating genetic heterogeneity within this disorder are described utilizing cultured fibroblasts from 11 different patients. Fibroblasts from I-cell disease (ICD) and from five different lysosomal storage diseases with single structural gene enzyme deficiencies were fused in different combinations, and fractions enriched for multinucleated heterokaryons were isolated and tested for acid hydrolase activity and electrophoretic mobility. In fusions of ICD fibroblasts and various single lysosomal enzyme-deficient fibroblasts, the activity of the deficient enzyme and of the other ICD hydrolases were restored, demonstrating that ICD is not a lysosomal enzyme structural gene defect and that the ICD defect, and not just the single enzyme deficiency, is corrected. In fusions involving only I-cell fibroblasts, at least two complementation groups were identified by the recovery of activities of all lysosomal enzymes tested in heterokaryons. These results demonstrate the existence of genetic heterogeneity within the disorder and suggest that different mutations can result in the I-cell clinical and biochemical phenotype. The data support an altered post-translational processing of lysosomal enzymes as the cause of ICD and suggest that at least two genes participate in this pathway.

Effect of chronic iodide supplements on thyroid secretion: the role of lysosomes.

The role of thyroid lysosomes in hormonal secretion has been studied in rats fed a low iodine diet and receiving chronic increasing iodide supplements. Thyroid secretion was assessed by plasma TSH, T3, and T4 levels and by the concentration and degree of iodination of thyroglobulin (Tgb). Lysosomal activity was evaluated by the total activity of four lysosomal acid hydrolases and by the integrity and fragility of the lysosomal membrane, which were assessed by the latency of acid phosphatase activity and the susceptibility of the particles to physicochemical manipulations. In glands from animals on a low iodine intake and under chronic TSH hyperstimulation, the integrity of the lysosomes was modified (increased free, but sedimentable, acid phosphatase activity), and the lysosomal membrane cohesion was high (increased resistance to freezingthawing and acid autolysis). In animals receiving increasing physiological iodide supplements, plasma TSH levels were normal, and the size of the intrathyroidal iodine pool autoregulated thyroid secretion; in spite of the increased concentration and iodine content of Tgb, the plasma levels of T3 and T4 remained fairly constant. The total activities of the four lysosomal enzymes tested increased gradually with the iodide supply. The integrity and stability of the lysosomes were not modified. Iodine at high pharmacological doses was not a lysosome stabilizer but induced a labilization of the lysosomal membrane. We suggest that lysosomes may participate in the autoregulation of thyroid secretion by the intrathyroidal iodine pool; the synthesis of new lysosomal hydrolases is induced, which may gradually be involved in the degradation of cellular constituents rather than in the hydrolysis of extracellular Tgb.

Serum activities of lysosomal enzymes in patients with liver cell carcinoma.

Serum activities of two lysosomal enzymes, beta-glucuronidase and acid phosphatase, were estimated in 66 patients with liver cell carcinoma, 10 with secondary liver cancer, 14 with cirrhosis of the liver, and 9 normal controls. A substantial increase in the enzyme activities was found in patients with liver cell carcinoma but not in those with secondary liver cancer. The degree of the enzyme elevations paralleled the stage of hepatoma. Although the serum activities of both enzymes were also elevated in patients with liver cirrhosis, the elevations were significantly higher in hepatoma than in liver cirrhosis. Possible mechanisms for the elevation of serum lysosomal enzyme activities in hepatoma are discussed, but further studies are necessary to elucidate the biological and clinicopathological significance of estimating serum lysosomal acid hydrolases in patients with primary liver cell carcinoma.