Warning: mkdir(): Permission denied in /home/virtual/lib/view_data.php on line 81
Warning: fopen(upload/ip_log/ip_log_2024-04.txt): failed to open stream: No such file or directory in /home/virtual/lib/view_data.php on line 83
Warning: fwrite() expects parameter 1 to be resource, boolean given in /home/virtual/lib/view_data.php on line 84 Characterization of a peroxidase in excretory-secretory product of adult Paragonimus westermani
Characterization of a peroxidase in excretory-secretory product of adult Paragonimus westermani
Y B Chung,1Y Kong,1S Y Cho,*1S Y Kang,1B C Choi,2 and H S Lee2
1Department of Parasitology, College of Medicine, Chung-Ang University, Seoul 156-756, Korea.
2Department of Biochemistry, College of Medicine, Chung-Ang University, Seoul 156-756, Korea.
Received June 30, 1993; Accepted July 21, 1993.
Abstract
When activity of peroxidase in adult Paragonimus westermani was monitored using o-dianisidine and H2O2 as substrates, its specific activity was 1.5 times higher in excretory-secretory product (ESP) than in crude extract. The enzyme was purified by two purification steps of Sephacryl S-300 Superfine gel permeation and DEAE-Trisacryl M anion exchange chromatographies. Its activity increased 16.9 fold with 32.3% recovery. The enzyme was inhibited totally by 1 millimoles of dithiothreitol (DTT), 2-mercaptoethanol and azide. Molecular mass was 16 kDa in reducing SDS-polyacrylamide gel electrophoresis (PAGE) or 19 kDa in TSK-Blue gel filtration high performance liquid chromatography (HPLC), respectively. Special staining for peroxidase by diaminobenzidine on SDS-PAGE confirmed the activity. The peroxidase was less reactive to a paragonimiasis serum when observed by SDS-PAGE/immunoblot. In addition, specific activities of superoxide dismutase (SOD) and catalase were also identified in the ESP. High activities of these antioxidant enzymes in ESP indicate that they are parts of defense mechanisms against reactive oxygen intermediates from host.
Figures
Fig. 1 Elution profile of ESP of adult P. westermani in a Sephacryl S-300 Superfine gel permeation chromatography (1.6 ×70 cm). The column was equilibrated with 0.01 M PBS (pH 7.4 containing 0.1 mM EDTA and eluted with the same buffer at a flow rate of 20nl/hour·cm2. Allocation of 1.8 ml were assayed for enzyme activities. • : Protein content,. Inverted arrowheads indicate the respective enzymes. Bar (-) indicates the pooled fraction of respective enzyme. ▪ : SOD activity, ▵ : Catalase activity, □ : Peroxidase activity.
Fig. 2 Elution profile of peroxidase in ESP of adutl P. westermani on DEAE-Trisacryl M anion exchange chromatography. Partially clarified enzyme solution containing high activity of peroxidase was applied to a 1.6 × 5 cm sized DEAE-Trisaryl M columnand eluted through 1.02 M Tris-HCl buffer (pH 7.2) containing 0, 0.02, 0.05. 0.1 ir 0.2 M stepwise salt concentration. activity, inverted arrow indicate the salt gradient. Bar (-) indicates the pooled fraction containing high peroxidase activity.
Fig. 3 Determination of molecular mass of perocidase by TSK-Blue analytical gel filtration HPLC. Ten microgram of the enzyme was eluted through the column at a flow rate of 1 ml/minute. Standard proteins used were alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa) and cytochrome c (15.4 kDa). Inverted arrow (↓) indicates the retention time of the peroxidase.
Fig. 4 SDS-PAGE findings of the purified peroxidase on 7.5~15% gel in reducing condition. Throughout the electrophoresis, constant current of 30 mA was supplied. The gel was stained with Coomassie blue R-250 and destained with methanol/acetic acid solution. Mr: Molecular mass in kDa, Lane 1 showing the crude ESP. Lane 2 exhibits Sephacryl S-300 filtered fraction and lane 3 demonstrates the purified peroxidase.
Fig. 5 Finding of the purified enzyme in special staining for peroxidase. Separating gel of 10~15% was used for portein separation in reducing condition. After SDS-PAGE, the gel was soaked in 0.01 M potassium phosphate buffer (pH 7.2) containing 2 mM o-dianisidine 2 mM 3,3-diaminobenzidine and stained with 0.1 mM H2O2 and Cr: crude extract of adult P. westermani, P: Purified peroxidase from the ESP.
Fig. 6 SDS-PAGE/immunoblot using a confirmed patient serum to the ESP, adult worm extract and the purified peroxidase showing a typical finding. After transfer the resolved proteins to nitrocellulose paper by electrophoresis at 100 V for 2 hours at 4℃, a patient serum, diluted at 1:150 in PBS/T, was reacted overnight as a probe. M2: Molecular mass in kDa, Cr: Crude ESP, P: Purified peroxidase, A: Whole worm extract of adult P. westermani.
Tables
Table 1 Comparison of activities of peroxidase, SOD and catalase in ESP and crude extract of adult P. westermani
Table 2 Purification of peroxidase in ESP of adult P. westermani
Table 3 Effect of inhibitors on peroxidase activity in ESP of adult P. westermani
References
1.
Aebi H. Methods of enzymatic analysis 1974;2:674–678.
2.
Barrett J. Peroxide metabolism in the liver fluke, Fasciola hepatica. J Parasitol 1980;66(4):697.
3.
Callahan HL, Crouch RK, James ER. Helminth anti-oxidant enzymes: a protective mechanism against host oxidants?. Parasitol Today 1988;4(8):218–225.
4.
Chung YB, Lee HS, Song CY, Cho SY. Activities of scavenging enzymes of oxygen radicals in early maturation stages of Paragonimus westermani. Korean J Parasitol 1992;30(4):355–358.
5.
Chung YB, Song CY, Lee HS, Kong Y, Cho SY. Purification and characterization of a Cu, Zn-superoxide dismutase from adult Paragonimus westermani. Korean J Parasitol 1991;29(3):259–266.
6.
Connors VA, Yoshino TP. In vitro effect of larval Schistosoma mansoni excretory-secretory products on phagocytosis-stimulated superoxide production in hemocytes from Biomphalaria glabrata. J Parasitol 1990;76(6):895–902.
7.
Harris ED. Regulation of antioxidant enzymes. FASEB J 1992;6(9):2675–2683.
8.
Henkle KJ, Liebau E, Muller S, Bergmann B, Walter RD. Characterization and molecular cloning of a Cu/Zn superoxide dismutase from the human parasite Onchocerca volvulus. Infect Immun 1991;59(6):2063–2069.
9.
Kazura JW, Meshnick SR. Scavenger enzymes and resistance to oxygen mediated damage in Trichinella spiralis. Mol Biochem Parasitol 1984;10(1):1–10.
10.
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227(5259):680–685.
11.
Leid RW, Suquet CM. A superoxide dismutase of metacestodes of Taenia taeniaeformis. Mol Biochem Parasitol 1986;18(3):301–311.
12.
Lightowlers MW, Rickard MD. Excretory-secretory products of helminth parasites: effects on host immune responses. Parasitology 1988;96 Suppl:S123–S166.
13.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193(1):265–275.
14.
Marikovsky M, Arnon R, Fishelson Z. Proteases secreted by transforming schistosomula of Schistosoma mansoni promote resistance to killing by complement. J Immunol 1988;141(1):273–278.
15.
Rivera Marrero CA, Santiago N, Hillyer GV. Evaluation of immunodiagnostic antigens in the excretory-secretory products of Fasciola hepatica. J Parasitol 1988;74(4):646–652.
16.
McCord JM, Fridovich I. The reduction of cytochrome c by milk xanthine oxidase. J Biol Chem 1968;243(21):5753–5760.
17.
Misra HP, Fridovich I. Superoxide dismutase and peroxidase: a positive activity stain applicable to polyacrylamide gel electropherograms. Arch Biochem Biophys 1977;183(2):511–515.
18.
Misra HP, Fridovich I. Inhibition of superoxide dismutases by azide. Arch Biochem Biophys 1978;189(2):317–322.
19.
Mkoji GM, Smith JM, Prichard RK. Antioxidant systems in Schistosoma mansoni: evidence for their role in protection of the adult worms against oxidant killing. Int J Parasitol 1988;18(5):667–673.
20.
Murray HW. Interaction of Leishmania with a macrophage cell line. Correlation between intracellular killing and the generation of oxygen intermediates. J Exp Med 1981;153(6):1690–1695.
21.
Orido Y. Development of the excretory bladder of the lung fluke Paragonimus ohirai (Trematoda: Troglotrematidae). J Parasitol 1990;76(2):205–211.
22.
Paul JM, et al. Int J Parasitol 1980;10:121–124.
23.
Poetter J, et al. Methods of enzymatic analysis 1984;3:286–293.
24.
Ys Shim,et al. Seminars Rep Med 1991;12:35–45.
25.
Simurda MC, van Keulen H, Rekosh DM, LoVerde PT. Schistosoma mansoni: identification and analysis of an mRNA and a gene encoding superoxide dismutase (Cu/Zn). Exp Parasitol 1988;67(1):73–84.
26.
Song CY, Dresden MH. Partial purification and characterization of cysteine proteinases from various developmental stages of Paragonimus westermani. Comp Biochem Physiol B 1990;95(3):473–476.
27.
Tsang VC, Peralta JM, Simons AR. Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis. Methods Enzymol 1983;92:377–391.
PDF Links
Full text via DOI
Download Citation 
