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.
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)
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