• Toxicological Research
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• v.17
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• pp.97-104
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• 2001

Three types of proteases (MEF-1, MEF-2 and MEF-3) were purified from the egg cases of Ten-odera sinensis using ammonium sulfate fractionation, gel filtration on Bio-Gel P-60 and affinity chromatography on DEAE Affi-Gel blue gel. The proteases were assessed homogeneous by SDS-polyacrylamide gel electrophoresis and have molecular weight of 31,500, 32,900 and 35,600 Da, respectively. The N-terminal regions of the primary structure were compared and they were found to be different each other. MEFs readily digested the $A\alpha$ - and B$\beta$-chains of fibrinogen and more slowly the ${\gamma}$-chain. The action of the enzymes resulted in extensive hydrolysis of fibrinogen and fibrin, releasing a variety of fibrinopeptides. MEF-1 was inactivated by Cu$^{2+}$ and Zn$^{2+}$ and inhibited by PMSF and chymostatin. MEF-2 was inhibited by PMSF, TLCK. soybean trypsin inhibitor. MEF-3 was only inhibited by PMSF and chymostatin. Antiplasmin was not sensitive to MEF-1 but antithrombin III inhibited the enzymatic activity qf MEF-1. MEF-2 specifically bound to anti plasmin Among the chromogenic protease substrates, the most sensitive one to the hydrolysis of MEFs was benzoyl-Phe-Val-Arg-p-nitroanilide with maximal activity at pH 7.0 and 3$0^{\circ}C$. MEF-1 preferentially cleaved the oxidized B-chain of insulin between Leu15 and Tyr16. In contrast, MEF-2 specifically cleaved the peptide bond between Arg23 and Gly24. D-dimer concentrations increased on incubation of cross-linked fibrin with MEF-1, indicating the enzyme has a strong fibrinolytic activity.ity.

• Clinical and Experimental Reproductive Medicine
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• v.21 no.1
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• pp.89-97
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• 1994

These studies were carried out to investigate the properties of H-Y antigen purified by immunoaffinity chromatography using monoclonal H-Y antibody. Immunoaffinity column was prepared by the coupling of monoclonal antibody to the Aminolink Coupling Gel. Murine testis supernatant was applied onto the column and eluted by O.lM glycine-HCl buffer and 31${\mu}g$ of H-Y Ag was eluted from one testis. Purified H-Y Ag strongly reacted with Con A and lentil from 6 different kinds of lectins tested, which may indicate that sugar moiety of H-Y Ag is composed of glucose, mannose and their derivatives. Con A-sepharose affinity column was used to purified H-Y Ag based on that H-Y Ag is glycoprotein. The fraction eluted by 0.2M Me-${\alpha}$-D-mannoside from the column loaded with murine testis supernatant was identified to be H-Y Ag by dot blot test. Molecular weight of the purified H-Y Ag was estimated by Sepharose G-75 gel filtration and SDS-PAGE, and showing that it was about 67,000 dalton. In fluorescence test, the ratio of XY embryos and XX embryos was 1:1.

• BMB Reports
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• v.38 no.5
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• pp.526-532
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• 2005

A lectin with in-vitro anticancer activity against established human cancer cell lines has been purified by affinity chromatography on asialofetuin-linked amino activated silica beads from the tubers of Arisaema tortuosum, popularly known as Himalayan Cobra lily, a monocot plant from the family Araceae. The bound Arisaema tortuosum lectin (ATL) was eluted with glycine-HCl buffer, pH 2.5. ATL was effectively inhibited by asialofetuin, a complex desialylated serum glycoprotein as well as by N-acetyl-D-lactosamine, a disaccharide. It gave a single band corresponding to a subunit molecular weight of 13.5 kDa in SDS-PAGE, pH 8.8 both under reducing and non reducing conditions. When subjected to gel-filtration on Biogel P-200, it was found to have a molecular weight of 54 kDa, suggesting a homotetramer structure, in which individual polypeptides are not bound to each other with disulfide bonds. ATL is a glycoprotein with 0.9% carbohydrate content, stable up to $55^{\circ}C$ and at pH 2 to 10. The lectin had no requirement for divalent metal ions i.e. $Ca^{2+}$ and $Mn^{2+}$ for its activity. However, as reported for other monocot lectins, ATL gave multiple bands in isoelectric focusing and Native PAGE, pH 8.3. The lectin was found to inhibit in vitro proliferation of human cancer cell lines HT29, SiHa and OVCAR-5.

• BMB Reports
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• v.36 no.2
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• pp.159-166
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• 2003

Cytoplasmic $\alpha$-glycerol-3-phosphate dehydrogenase from fruit-bat-breast muscle was purified by ion-exchange and affinity chromatography. The specific activity of the purified enzyme was approximately 120 units/mg of protein. The apparent molecular weight of the native enzyme, as determined by gel filtration on Sephadex G-100 was $59,500{\pm}650$ daltons; its subunit size was estimated to be $35,700{\pm}140$ by SDS-polyacrylamide gel electrophoresis. The true Michaelis-Menten constants for all substrates at pH 7.5 were $3.9{\pm}0.7\;mM$, $0.65{\pm}0.05\;mM$, $0.26{\pm}0.06\;mM$, and $0.005{\pm}0.0004\;mM$ for L-glycerol-3-phosphate, $NAD^+$, DHAP, and NADH, respectively. The true Michaelis-Menten constants at pH 10.0 were $2.30{\pm}0.21\;mM$ and $0.20{\pm}0.01\;mM$ for L-glycerol-3-phosphate and $NAD^+$, respectively. The turnover number, $k_{cat}$, of the forward reaction was $1.9{\pm}0.2{\times}10^4\;s^{-1}$. The treatment of the enzyme with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) under denaturing conditions indicated that there were a total of eight cysteine residues, while only two of these residues were reactive towards DTNB in the native enzyme. The overall results of the in vitro experiments suggest that $\alpha$-glycerol-3-phosphate dehydrogenase of the fruit bat preferentially catalyses the reduction of dihydroxyacetone phosphate to glycerol-3-phosphate.

• Archives of Pharmacal Research
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• v.21 no.2
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• pp.128-134
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• 1998

Nitric oxide synthase, NOS (EC.1.14.13.39), was purified from bovine pancreas over 5,500-fold with a 7.6% yield using 30% ammonium sulfate precipitation, and $2^1$,$5^1$-ADP-agarose and calmodulin-agarose affinity chromatography. The purified bovine pancreatic NOS (bpNOS) showed a single band on SDS-PAGE corresponding to an apparent molecular mass of 160 kDa, whereas it was 320 kDa on non-denaturating gel-filtration. This indicated a homodimeric nature of the enzyme. The specific activity of the purified bpNOS was 31.67 nmol L-citrulline fored/mtn/mg protein and an apparent $K\textrm{m}$ for L-arginine was 15.72 $\mu\textrm{M}$, The enzyme activity was dependent on $Ca^{2+}$ and calmodulin, and to a lesser extent on NADPH, FAD and FMN. $H_4B$ was not required as a cofactor for the activity. In an inhibition experiment with L-arginine analogues, $N^G$-nitro-L-arginine (NNA) had the most potent inhibitory effect on bpNOS, and $N^{G}$, $N^{G1}$-dimethyl-L-arginine (symmetric; sDMA) did not have any inhibitory effect. Immunohistochemical analysis of the bovine pancreas using brain type NOS antibody (anti-bNOS antibody) revealed that acinar cells showed strong immunoreactivity against the antibody.

• Korean Journal of Veterinary Research
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• v.40 no.1
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• pp.86-93
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• 2000

Paratuberculosis (Johne's disease), a chronic enteritis produced by Mycobacterium paratuberculosis, affects a large proportion of ruminants in all continents and causes important economic losses. The identification of well-characterized and species-specific components of M paratuberculosis would provide the means to improve the specificity and sensitivity of immunodiagnostic assays for Johne's disease. The aims of this study were to express the recombinant C-terminal of 34kDa protein (rC34P) of M paratuberculosis in E coli and to investigate the effectiveness of this protein in detecting antibodies to the native protein in sera from paratuberculosis infected cattle. The C-terminal of the gene encoding the 34kDa protein was amplified by polymerase chain reaction from the chromosomal DNA of M paratuberculosis (ATCC 19698) and cloned into vector pGEX-4T-2. Then, cloned plasmid was transformed into E coli DH5${\alpha}$ and the rC34P was overexpressed. The rC34P was purified by affinity chromatography and gel filtration. The rC34P was examined antigenicity by Western blot. The rC34P was reactive with culture positive bovine serum and hyperimmune rabbit anti-M paratuberculosis serum but was not reactive with culture negative bovine serum and tuberculin positive bovine serum in Western blot. In conclusion, the rC34P produced in this study is expected as a useful candidate for antigen in serological diagnosis of Johne's disease.

• Fisheries and Aquatic Sciences
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• v.1 no.2
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• pp.201-208
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• 1998

A protease, which had no tryptic and chymotryptic activity, was purified from the hepatopancreas of shrimp, P. japonicus, through ammonium sulfate fractionation, Q­Sepharose ionic exchange, benzamidine Sepharose 6B affinity, and Sephacryl S-100 gel chromatography. Molecular weight (M.W.) of the protease was estimated to be 24 kDa by gel filtration and showed a single peptide band by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protease had a low ratio of acidic to basic amino acids, which is different with pro teases from marine animals. The enzyme was partially inhibited by benzamidine, tosyl-L-lysine chioromethyl ketone (TLCK), phenylmethylsulfonyl fluoride (PMSF), soybean trypsin inhibitor (SBTI), and pepstatin. The enzyme did not have any activity against benzoyl-D,L-arginine p-nitroanilide (BAPNA) or benzoyl-L-tyrosine ethyl ester (BTEE) which is a specific substrate of trypsin and chymotrypsin, respectively. However, the enzyme showed activity forward N-CBZ-L-tyrosine p-nitrophenyl ester (CBZ-Tyr-pNE), N­CBZ-L-tryptophan p-nitrophenyl ester (CBZ-Trp-pNE), and N-CBZ-L-proline p-nitrophenyl ester (CBZ-Pro-pNE). The protease did not showed tryptic and chymotryptic activity, which was not reported in shrimp hepatopancreas.

• BMB Reports
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• v.35 no.2
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• pp.206-212
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• 2002

The NS2B-NS3(pro) polyprotein segment from the dengue virus serotype 2 strain 16681 was purified from overexpressing E. coli by metal chelate affinity chromatography and gel filtration. Enzymatic activity of the refolded NS2B-NS3(pro) protease complex was determined in vitro with dansyl-labeled peptide substrates, based upon native dengue virus type 2 cleavage sites. The 12mer substrate peptides and the cleavage products could be separated by reversed-phase HPLC, and were identified by UV and fluorescence detection. All of the peptide substrates (representing the DEN polyprotein junction sequences at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 sites) were cleaved by the recombinant protease NS2B-NS3(pro). No cleavage was observed with an enzymatically inactive S135A mutant of the NS3 protein, or with a modified substrate peptide of the NS3/NS4A polyprotein site that contained a K2093A substitution. Enzymatic activity was dependent on the salt concentration. A 50% decrease of activity was observed in the presence of 0.1M sodium chloride. Our results show that the NS3 protease activity of the refolded NS2B-NS3(pro) protein can be assayed in vitro with high specificity by using cleavage-junction derived peptide substrates.

• Archives of Pharmacal Research
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• v.28 no.7
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• pp.816-822
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• 2005

Catharsius protease-2 (CPM-2) was isolated from the body of dung beetles, Catharsius molossus, using a three step purification process (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and affinity chromatography on DEAE Affi-Gel blue). The purified CPM-2, having a molecular weight of 24 kDa, was assessed homogeneously by SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of CPM-2 was composed of X Val Gin Asp Phe Val Glu Glu lie Leu. CPM-2 was inactivated by $Cu^{2+}\;and\;Zn^{2+}$ and strongly inhibited by typical serine proteinase inhibitors such as TLCK, soybean trypsin inhibitor, aprotinin, benzamidine, and ${\alpha}_1$-antitrypsin. However, EDTA, EGTA, cysteine, $\beta$-mercaptoethanol, E64, and elastatinal had little effect on enzyme activity. In addition, antiplasmin and antithrombin III were not sensitive to CPM-2. Based on the results of a fibrinolytic activity test, CPM-2 readily cleaved $A{\alpha}-$ and $B{\beta}$-chains of fibrinogen and fibrin, and y-chain of fibrinogen more slowly. The nonspecific action of the enzyme resulted in extensive hydrolysis, releasing a variety of fibrinopeptides of fibrinogen and fibrin. Polyclonal antibodies of CPM-2 were reactive to the native form of antigen. The ELISA was applied to detect quantities, in nanograms, of the antigen in CPM-2 protein.

• Journal of Microbiology and Biotechnology
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• v.7 no.2
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• pp.107-113
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• 1997

A strain producing a high amount of chitinase was isolated from soil, identified as Pseudomonas sp., and tentatively named Pseudomonas sp. YHS-A2. An extracellular chitinase of Pseudomonas sp. YHS-A2 was purified according to the procedure of ammonium sulfate saturation, affinity adsorption, Sephadex G-100 gel filtration and Phenyl-sepharose CL-4B hydrophobic interaction column chromatography. The molecular weight of the purified enzyme was estimated to be 55 kDa on SDS-PAGE was confirmed by active staining. Optimal pH and temperature of the enzyme are pH 7.0 and $50^{\circ}C$, respectively, and the enzyme is stable between pH 5.0 and 8.0 and below $50^{\circ}C$. The main products of colloidal chitin by the chitinase were N-acetyl-D-glucosamine and N,N'-diacetylchitobiose both of which were detected by HPLC analysis. The enzyme is supposed to be a random-type endochitinase which can degrade any position of ${\beta}$-l,4-linkages of chitin and chitooligosaccharides. The chitinase inhibited the growth of some phytopathogenic fungi, Fusarium oxysporum, Botrytis cineria, and Mucor rouxii and these antifungal effects were thought to be due to the characteristics of endochitinase.