• Natural Product Sciences
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• v.13 no.4
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• pp.289-294
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• 2007

Ethanolic extract (50% v/v) and alkaloid fraction of Tylophora indica leaves were examined for lysosomal enzyme inhibitory activity in adjuvant-induced arthritic rats. The alkaloid fraction showed statistically significant inhibition of arthritic lesions (p < 0.05) from day 18, (p < 0.025) from day 20 and (p < 0.001) from day 21 onwards in the adjuvant-induced arthritis, which was comparable to the response of standard drug Indomethacin. The ethanolic extract was less significant than the alkaloidal fraction in inhibition of arthritis. Alkaloid fraction showed significant (p < 0.001) inhibitory effect on the lysosomal enzyme activities in adjuvantinduced arthritic rats. It also significantly prevented decrease in collagen levels and synovial damage observed during arthritis and also inhibited increase in urinary excretion levels of collagen degradation products like hydroxyproline, hexosamine, hexuronic acid, etc. Both ethanolic extract as well as the alkaloid fraction, however, did not show any significant activity in normal nonarthritic rats. The ethanolic extract and the alkaloid fraction may thus be able to inhibit the progress of inflammation and inhibit the destructive activity of lysosomal enzymes on structural macromolecules like collagen etc. in the synovial capsule in joints during arthritic states. They may thus prevent synovial damage observed during arthritis.

• BMB Reports
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• v.29 no.3
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• pp.253-260
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• 1996

An enzyme that hydrolyzes flavin-adenine dinucleotide (FAD) was found to be present in rat liver lysosomal membrane prepared from Triton WR-1339 filled lysosomes (tritosomes) purified by flotation on sucrose. This FAD phosphohydrolase (FADase) exhibited optimal activity at pH 8.5 and had an apparent Km of approximately 3.3 mM. The activity was decreased 50~70% by dialysis against EDTA and this was restored by $Zn^{2+}$, $Mg^{+2}$, $Hg^{+2}$, and $Ca^{+2}$ ions inhibited the enzyme, but $F^-$ and molybdate had no effect. The enzyme was also inhibited by p-chloromercuribenzoate (pCMB), reduced glutathione and other thiols, cyanide, and ascorbate. The presence of ATP, ADP, AMP. ${\alpha}-{\beta}-methylene$ ATP, AMP-p-nitrophenyl phosphate (PNP), GMP, and coenzyme A (CoA) decreased the activity on FAD, but pyrimidine nucleotides, adenosine, adenine, or $NAD^+$ were without effect. Phosphate stimulated the activity slightly. FAD phosphohydrolase activity was separated from ATPase and inorganic pyrophosphatase activities by solubilization with detergents and polyacrylamide gel electrophoresis and by linear sucrose density gradient centrifugation suggesting that the enzyme is different from ATPase, inorganic pyrophosphatase, and soluble lysosomal FAD pyrophosphatase. Paper chromatography showed that FAD was hydrolyzed to flavin mononucleotide (FMN) and AMP which were further hydrolyzed to riboflavin and AMP by phosphatases known to be present in lysosomal membranes. Incubation of the intact Iysosomes with pronase showed that the active site of FAD phosphohydrolase must be oriented to the cytosol. The FAD hydrolyzing activity was detected in Golgi, microsome, and plasma membrane, but not in mitochondria or soluble lysosomal preparations.

• The Korean Journal of Pharmacology
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• v.25 no.1
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• pp.75-85
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• 1989

In opsonized zymosan activated PMN leukocytes, N-ethylamleiamide and $Hg^{++}$, penetrable sulfhydryl group inhibitors, inhibited superoxide generation, NADPH oxidase activity and lysosomal enzyme (lactic dehydrogenase and ${\beta}-glucuronidase$) secretion. P-Chloromercuribenzoic acid and p-chloromercuribenzenesulfonic acid, surface sulfhydryl group inhibitors did not affect superoxide generation but effectively inhibited both NADPH oxidase activity and lysosomal enzyme secretion. During phagocytosis, contents of surface and soluble sulfhydryl groups were gradually decreased with increasing incubation times. N-ethylmaleiamide and $Hg^{++}$ caused a loss of both surface and soluble sulfhydryl groups. P-Chloromercuribenzoic acid and p-chloromercuribenzenesulfonic acid significantly decreased the surface sulfhydryl content but did not after soluble sulfhydryl groups. Cysteine and mercaptopropionylglycine inhibited superoxide generation and lysosomal enzyme secretion. Glutathione had no effect on superoxide generation but remarkably inhibited lactic dehydrogenase release. Suppression of superoxide generation by N-ethylmaleiamide was reversed by cysteine and mercaptopropionyl-glycine but not by glutathione. Inactivation of NADPH oxidase by N-ethylmaleiamide was prevented by glutathione, cysteine or mercaptopropionylglycine. Stimulated superoxide generaion by carbachol was completely abolished by N-ethylrnaleiamide and antagonized by atropine. Thus, the expression of PMN leukocyte response to external stimuli may be associated with the change of sulfhydryl groups content. It is suggested that lysosomal enzyme secretion is influenced by both surface and soluble sulfhydryl groups, whereas superoxide generation by intracellular soluble sulfhydryl groups.

• Journal of Microbiology and Biotechnology
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• v.27 no.2
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• pp.372-379
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• 2017

The transport of lysosomal enzymes into the lysosomes depends on the phosphorylation of their chains and the binding of the phosphorylated residues to mannose-6-phosphate receptors. The efficiency of separation depends more on the phosphodiesterases (PDEs) than on the activity of the phosphorylation of mannose residues and can be determined in vitro. PDEs play important roles in regulation of the activation of lysosomes. The expression of proteins was confirmed by western blotting. All PDE4 series protein expression was reduced in high concentrations of rolipram. As a result of observing the fluorescence intensity after rolipram treatment, the lysosomal enzyme was activated at low concentrations and suppressed at high concentrations. High concentrations of rolipram recovered the original function. Antimicrobial activity was not shown in either 10 or $100{\mu}M$ concentrations of rolipram in treated HeLa cells in vitro. However, the higher anticancer activity at lower rolipram concentration was shown in lysosomal enzyme treated with $10{\mu}M$ of rolipram. The anticancer activity was confirmed through cathepsin B and D assay. Tranfection allowed examination of the relationship between PDE4 and lysosomal activity in more detail. Protein expression was confirmed to be reduced. Fluorescence intensity showed decreased activity of lysosomes and ROS in cells transfected with the antisense sequences of PDE4 A, B, C, and D. PDE4A showed anticancer activity, whereas lysosome from cells transfected with the antisense sequences of PDE4 B, C, and D had decreased anticancer activity. These results showed the PDE4 A, B, C, and D are conjunctly related with lysosomal activity.

• Journal of Life Science
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• v.19 no.11
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• pp.1617-1622
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• 2009

Optium conditions for the production of mycelia and extracellular polysaccharide (EXPS) from submerged mycelial culture of Inonotus obliquus and their immunomodulating activities were investigated. The optmium production of mycelia and EXPS from I. obliquus was observed in mushroom complete medium (MCM). The optimum pH, temperature, and agitation speed for the production of mycelia and EXPS were 5.5, $25^{\circ}C$, and 150 rpm, respectively. The culture period for maximum production of mycelia (10.89 g/l) and EXPS (1.25 g/l) in shake flask cultivation was 11 days. The anticomplementary activity of intracellular polysaccharide (INPS) and EXPS form I. obliquus increased in a dose-dependent manner. Lysosomal enzyme activity of EXPS and INPS increased by 2.0- and 2.2-fold at $100{\mu}g/ml$ concentration, respectively, compared to the control group.

• Applied Biological Chemistry
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• v.20 no.2
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• pp.175-181
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• 1977

Lysosomal enzyme latency was demonstrated for hydrolases from porcine leukocyte by suspending sediment sfrom differential centrifugation in 0.125 to 0.250 M sucrose. Specific activities pH optima and activation energies were determined for hydrolases distributed in various sedimentation fractions and for enzymes solubilized by n-butyl alcohol extraction. Specific activities of the hydrolases revealed the heterogeneity of the Iysosomal fractions relative to enzyme content. pH optima identified the enzyme as acid hydrolases with optima for cathepsin D and aryl sulfatase also at pH 6.8. Activation energies of some hydrolases were low revealing that these enzymes could function efficiently during low temperature aging of meat.

• Journal of the Korean Society of Food Science and Nutrition
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• v.31 no.3
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• pp.527-533
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• 2002

In the screening of Korean traditional tea sources for the cellular lysosomal enzyme activity of peritoneal macrophage from mice, CT-0, a cold-water extract from Carthamus tinctorius L., showed the highest macro-phage-stimulating activity. CT-1-IIa-2-1, a purified macrophage-stimulation polysaccharide was obtained by a series of purification steps such as anion exchage chromatography with DEAE-Toyopearl 650M, gel permeation chromatography with Sepharose CL-6B, Sephacryl S-200, and HPLC with Superdex G-75. The molecular weight of homogeneous purified polysaccharide was estimated about 68 kDa. CT-1-IIa-2-1 consisted of xylose 27.44%, arabinose 16.14%, mannose 15.92% and glucose 14.47%. To measure acute toxicity, dose of 50, 100, 500, and 1000 mg/kg were intraperitoneally injected to ICR mice. The LD$\_$50/ was about 397 mg/kg.

• Journal of Microbiology and Biotechnology
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• v.31 no.1
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• pp.163-170
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• 2021

Enzyme replacement therapy for lysosomal storage diseases usually requires recombinant enzymes containing mannose-6-phosphate (M6P) glycans for cellular uptake and lysosomal targeting. For the first time, a strategy is established here for the in vitro mannosyl-phosphorylation of high-mannose type N-glycans that utilizes a recombinant Mnn14 protein derived from Saccharomyces cerevisiae. Among a series of N-terminal- or C-terminal-deleted recombinant Mnn14 proteins expressed in Pichia pastoris, rMnn1477-935 with deletion of N-terminal 76 amino acids spanning the transmembrane domain (46 amino acids) and part of the stem region (30 amino acids), showed the highest level of mannosyl-phosphorylation activity. The optimum reaction conditions for rMnn1477-935 were determined through enzyme assays with a high-mannose type N-glycan (Man8GlcNAc2) as a substrate. In addition, rMnn1477-935 was shown to mannosyl-phosphorylate high-mannose type N-glycans (Man7-9GlcNAc2) on recombinant human lysosomal alpha-glucosidase (rhGAA) with remarkably high efficiency. Moreover, the majority of the resulting mannosyl-phosphorylated glycans were bis-form which can be converted to bis-phosphorylated M6P glycans having a superior lysosomal targeting capability. An in vitro N-glycan mannosyl-phosphorylation reaction using rMnn1477-935 will provide a flexible and straightforward method to increase the M6P glycan content for the generation of "Biobetter" therapeutic enzymes.

• Korean Journal of Food Science and Technology
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• v.22 no.5
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• pp.569-574
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• 1990

The influence of NaCl and pH on degradation of chicken breast muscle myofibrillar proteins by porcine leukocyte lysosomal proteinases was investigated. The degradation reactions were carried out at $38^{\circ}C$ for 24hours under different conditions. The degradation of myofibrillar proteins by leukocyte lysosomal enzymes at various pH values was limited to partial hydrolysis. Reactions at higher pH values resulted in lower molecular weight degradation products while reactions at lower pH resulted in higher molecular weight degradation products. When NaCl was added into the reaction mixture, enzyme activities of degradation were increased at all pH values studied, as evidenced by NPN-analysis and SDS-PAGE. More severe degradation was observed with higher salt concentration. The concentration of 0.5M NaCl in the reaction mixture gave more degradation of myosin heavy chain by enzyme than that of 0.1M NaCl.

• Fisheries and Aquatic Sciences
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• v.5 no.4
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• pp.263-270
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• 2002

The Pacific oysters, Crassostrea gigas, were stressed with different concentrations of benzo(a) pyrene and depurated to determine the hemocyte lysosomal membrane stability and hydrolytic enzymatic activity as a biomarker candidate to the chemical, using NRR (neutral red retention) and API ZYM System, respectively. The membrane damage measured as NRR decrease was significant with the increase of chemical concentration and exposure time (P<0.05), providing a possible tool for biomarker. Interestingly, the control showed intrinsic stress probably due to captive life in the laboratory, and a recovering trend was also found during the depuration. The benzo(a)pyrene-exposed oysters showed increased enzyme activities in alkaline phosphatase, esterase (C4), acid phosphatase, naphthol-AS-BI-phospho­hydrolase, $\beta$-galactosidase, $\beta$-glucuronidase, and N-acetyl- $\beta$-glucosaminidase. Of them, only two enzymes, acid phosphatase and alkaline phosphatase, showed some potential available for the generation of enzymatic biomarker in the oyster. The results are suggestive of the potential availability of the cellular and enzymatic properties as a biomarker. However, considering that a robust biomarker should be insensitive to natural stress coming from normal physiological variation, but sensitive to pollutants, a concept of intrinsic stress the animal possesses should be taken into consideration. This reflects the necessity of further research on the intrinsic stress affecting the cellular and enzymatic properties of the chemical­stressed oysters prior to using the data as a biomarker.