Characterization of proteases of <i>Toxoplasma gondii</i>

, Choi, W Y; , Nam, H W; , Youn, J H

doi:1989.27.3.161

Korean J Parasito > Volume 27(3):1989 > Article

Original Article


Korean J Parasitol. 1989 Sep;27(3):161-170. English. Published online Mar 20, 1994. http://dx.doi.org/10.3347/kjp.1989.27.3.161
Copyright © 1989 by The Korean Society for Parasitology


Characterization of proteases of Toxoplasma gondii
W Y Choi,H W Nam and J H Youn
Department of Parasitology, Catholic University Medical College, Seoul 137-701, Korea.


Abstract
The proteases of Toxoplasma gondii were purified partially and characterized for some biochemical properties including various chromatographic patterns, major catalytic classes, and conditions to promote the activity of these enzymes. When Toxoplasma extract was incubated with 3H-casein at various pH, peak hydrolysis of casein was observed at pH 6.0 and at pH 8.5. Proteases working at pH 6.0 and at pH 8.5 were purified partially by conventional methods of chromatographies of DE52 anion exchange, Sephadex G-200 gel permeation, and hydroxylapatite chromatography. Partially purified enzymes were tested by site-specific inhibitors and promotors. The protease working at pH 6.0 was inactivated by iodoacetamide with LD₅₀ of 10^-3 M and promoted by dithiothreitol, while the protease working at pH 8.5 was inhibited by phenylmethylsulfonyl fluoride with LD₅₀ of 10^-5 M and was promoted by ATP (excess ATP beyond 2 mM inhibited the activity reversely). The protease of pH 8.5 had the activity of ATPase which might exert the energy to its action. Therefore the former was referred to as a cysteinyl acid protease and the latter, ATP-dependent neutral serine protease.

Figures


	Fig. 1 Effects of pH on the caseinolytic activity of Toxoplasma extract. Proteolytic activity was tested at various pH with casein in 50mM citrate buffer from pH 4.0 to 6.5 (-□-), acetate buffer from pH 5.0 to 7.5 (-○-), and Tris buffer from pH 7.0 to 10.0(-△-). the effects of DTT(-■- and ATP (-▼-) was also tested with the above buffers.


	Fig. 2 Effects of protease inhibitors on Toxoplasma extract at pH 6.0(■) and pH 8.5(□).


	Fig. 3 DE52 chromatography of Toxoplasma extract. Toxoplasma crude extract was loaded on a DE52 column and eluted with salt gradient of 0 to 0.4 M NaCl in 10mM Tris buffer, pH 7.4. Salt concentration gradient was indicated by a triangle. (-□-, at pH 6.0; -○-, at pH 8.5; -■-, A280; and -, saved fractions)


	Fig. 4 Sephadex G-200 gel permeation chromatography of fractions of high caseinolytic activity at pH 6.0 from DE52 fractionation. (-□-, % hydrolysis of casein; -■-, A₂₈₀; and -, saved fractions)


	Fig. 5 Sephadex G-200 gel permeation chromatography of fractions of high caseinolytic activity at pH 8.5 from DE52 fractionation, Refer to the logend of Fig. 4.


	Fig. 6 Hydroxylapatite chromatography of pH 6.0 proteolytic fractions. Highly active fractions from Sephadex G-200 column were pooled, dialyzd against 10 mM phosphate buffer, pH 7.4, and loaded on a gydroxylapatite column as described in the text. Phosphate concentration gradient was indicated by a triangle. (-□-, % hydrolysis of casein; -■-, A₂₈₀; and -, saved fractions)


	Fig. 7 Hydroxylapatite chromatography of pH 8.5 proteolytic fractions Refer to the legend of Fig. 6.


	Fig. 8 Effects of inhibitors on the caseinolytic activity of pH 6.0 active fractions. (-□-, Pep A; -○-, EDTA; -■-, IAAl and -●-, PMSF more diluted by 10²)


	Fig. 9 Effects of inhibitors on the caseinolytic activity of pH 8.5 active fractions. Refer to the logend of Fig. 8.


	Fig. 10 Effects of various concentrations of ATP on the caseinolytic activity of neutral protease of pH 8.5. (-○-, % hydrolysis of casein)

References


1.	Alfieri SC, Shaw E, Zilberfarb V, Rabinovitch M. Leishmania amazonensis: involvement of cysteine proteinases in the killing of isolated amastigotes by L-leucine methyl ester. Exp Parasitol 1989;68(4):423–431.

2.	Anderson KP, Atlas E, Ahern MJ, Weisbrot IM. Central nervous system toxoplasmosis in homosexual men. Am J Med 1983;75(5):877–881.

3.	Asch HL, Dresden MH. Acidic thiol proteinase activity of Schistosoma mansoni egg extracts. J Parasitol 1979;65(4):543–549.

4.	Avila EE, Sánchez-Garza M, Calderón J. Entamoeba histolytica and E. invadens: sulfhydryl-dependent proteolytic activity. J Protozool 1985;32(1):163–166.

6.	Bernard F, Schrével J. Purification of a Plasmodium berghei neutral endopeptidase and its localization in merozoite. Mol Biochem Parasitol 1987;26(1-2):167–173.

7.	Choi WY, Nam HW, Yoo JE. Toxoplasmacidal effect of HL-60 cells differentiated by dimethylsulfoxide. Korean J Parasitol 1988;26(4):229–238.

8.	Choi WY, Yoo JE, Nam HW, Oh CY, Kim SW, Katakura K, Kobayashi A. Toxoplasma antibodies by indirect latex agglutination tests in zoo animals. Korean J Parasitol 1987;25(1):13–23.

9.	Coombs GH. Proteinases of Leishmania mexicana and other flagellate protozoa. Parasitology 1982;84(1):149–155.

*10.*	Coombs GH, North MJ. An analysis of the proteinases of Trichomonas vaginalis by polyacrylamide gel electrophoresis. Parasitology 1983;86(Pt 1):1–6.

*11.*	Dluzewski AR, Rangachari K, Gratzer WB, Wilson RJ. Inhibition of malarial invasion of red cells by chemical and immunochemical linking of spectrin molecules. Br J Haematol 1983;55(4):629–637.

*12.*	Dluzewski AR, Rangachari K, Wilson RJ, Gratzer WB. Plasmodium falciparum: protease inhibitors and inhibition of erythrocyte invasion. Exp Parasitol 1986;62(3):416–422.

*13.*	Friedman MJ, Fukuda M, Laine RA. Evidence for a malarial parasite interaction site on the major transmembrane protein of the human erythrocyte. Science 1985;228(4695):75–77.

*14.*	Gadasi H, Kobiler D. Entamoeba histolytica: correlation between virulence and content of proteolytic enzymes. Exp Parasitol 1983;55(1):105–110.

*15.*	Keene WE, Petitt MG, Allen S, McKerrow JH. The major neutral proteinase of Entamoeba histolytica. J Exp Med 1986;163(3):536–549.

*16.*	Krick JA, Remington JS. Toxoplasmosis in the adult--an overview. N Engl J Med 1978;298(10):550–553.

*17.*	Letch CA, Gibson W. Trypanosoma brucei: the peptidases of bloodstream trypanosomes. Exp Parasitol 1981;52(1):86–90.

*18.*	Levine ND. Tazonomy of Toxoplasma. J Protozool 1977;24(1):36–41.

*19.*	Lindmark DG. Giardia lamblia: localization of hydrolase activities in lysosome-like organelles of trophozoites. Exp Parasitol 1988;65(1):141–147.

*20.*	Luft BJ, Brooks RG, Conley FK, McCabe RE, Remington JS. Toxoplasmic encephalitis in patients with acquired immune deficiency syndrome. JAMA 1984;252(7):913–917.

*21.*	Lushbaugh WB, Hofbauer AF, Pittman FE. Proteinase activities of Entamoeba histolytica cytotoxin. Gastroenterology 1984;87(1):17–27.

*22.*	Lushbaugh WB, Hofbauer AF, Pittman FE. Entamoeba histolytica: purification of cathepsin B. Exp Parasitol 1985;59(3):328–336.

*23.*	Maki J, Furuhashi A, Yanagisawa T. The activity of acid proteases hydrolysing haemoglobin in parasitic helminths with special reference to interspecific and intraspecific distribution. Parasitology 1982;84(1):137–147.

*24.*	Marciano-Cabral F, Stanitski S, Radhakrishna V, Bradley SG. Characterization of a neutral aminoacyl-peptide hydrolase from Naegleria fowleri. J Protozool 1987;34(2):146–149.

*25.*	McDonald JK. An overview of protease specificity and catalytic mechanisms: aspects related to nomenclature and classification. Histochem J 1985;17(7):773–785.

*26.*	MacKeen LA, DiPeri C, Schwartz I. Reductive methylation of IF-3 and EFTu with [¹⁴C]formaldehyde and sodium cyanoborohydride. FEBS Lett 1979;101(2):387–390.

*27.*	Muñoz ML, Rojkind M, Calderon J, Tanimoto M, Arias-Negrete S, Martínez-Palomo A. Entamoeba histolytica: collagenolytic activity and virulence. J Protozool 1984;31(3):468–470.

*28.*	Perez-Montfort R, Ostoa-Saloma P, Velazquez-Medina L, Montfort I, Becker I. Catalytic classes of proteinases of Entamoeba histolytica. Mol Biochem Parasitol 1987;26(1-2):87–97.

*29.*	Pupkis MF, Coombs GH. Purification and characterization of proteolytic enzymes of Leishmania mexicana mexicana amastigotes and promastigotes. J Gen Microbiol 1984;130(9):2375–2383.

*30.*	Pupkis MF, Tetley L, Coombs GH. Leishmania mexicana: amastigote hydrolases in unusual lysosomes. Exp Parasitol 1986;62(1):29–39.

*31.*	Rabinovitch M, Zilberfarb V, Pouchelet M. Leishmania mexicana: destruction of isolated amastigotes by amino acid esters. Am J Trop Med Hyg 1987;36(2):288–293.

*32.*	Rangel HA, Araújo PM, Repka D, Costa MG. Trypanosoma cruzi: isolation and characterization of a proteinase. Exp Parasitol 1981;52(2):199–209.

*33.*	Rautenberg P, Schädler R, Reinwald E, Risse HJ. Study on a proteolytic enzyme from Trypanosoma congolense. Purification and some biochemical properties. Mol Cell Biochem 982 Sep;47(3):151–159.

*34.*	Schrevel J, et al. Biochem biophy Res Commun 1984;124:703–710.