An evaluation of glutathione transferase associated with Dichlorvos degradation in African palm weevil (Rynchophorus phoenicis) larva

Figures & data

Figure 1. Glutathione peroxidase activity of fat body (FB), gut (GT), and head (H) tissues of Rynchophorous phoenicis larva treated with varied concentration of DDVP solution.

Notes: Each value is the mean of five repetitions ± SD. Significant difference in the treated groups from their corresponding control was indicated by alphabets (a-f) at p < 0.05.
Key: Control-larva treated with acetone in normal saline; D0.20-larva treated with 0.20 µg g⁻¹ (w/v) DDVP; D0.30-larva treated with 0.30 µg g⁻¹ (w/v); DDVP D0.40-larva treated with 0.40 µg g⁻¹ (w/v); DDVP D0.50-larva treated with 0.50 µg g⁻¹ (w/v); DDVP D0.60-larva treated with 0.60 µg g⁻¹ (w/v) DDVP.

Figure 2. Glutathione reductase activity of fat body (FB), gut (GT), and head (H) tissues of Rynchophorous phoenicis larva treated with varied concentration of DDVP solution.

Note: Each value is the mean of five repetitions ± SD. Significant difference in the treated groups from their corresponding control was indicated by alphabets (a-f) at p < 0.05.
Key: Control-larva treated with acetone in normal saline; D0.20-larva treated with 0.20 µg g⁻¹ (w/v) DDVP; D0.30-larva treated with 0.30 µg g⁻¹ (w/v); DDVP D0.40-larva treated with 0.40 µg g⁻¹ (w/v); DDVP D0.50-larva treated with 0.50 µg g⁻¹ (w/v); DDVP D0.60-larva treated with 0.60 µg g⁻¹ (w/v) DDVP.

Figure 3. Glutathione transferase activity of fat body (FB), gut (GT), and head (H) tissues of Rynchophorous phoenicis larva treated with varied concentration of DDVP solution.

Note: Each value is the mean of five repetitions ± SD. Significant difference in the treated groups from their corresponding control was indicated by alphabets (a-f) at p < 0.05.
Key: Control-larva treated with acetone in normal saline; D0.20-larva treated with 0.20 µg g⁻¹ (w/v) DDVP; D0.30-larva treated with 0.30 µg g⁻¹ (w/v); DDVP D0.40-larva treated with 0.40 µg g⁻¹ (w/v); DDVP D0.50-larva treated with 0.50 µg g⁻¹ (w/v); DDVP D0.60-larva treated with 0.60 µg g⁻¹ (w/v) DDVP.

Figure 4. Time-course glutathione transferase activity (GST) in the gut tissue of Rynchophorous phoenicis larva.

Note: Each value is the mean of three repetitions ± SD. Experiment was conducted at room temperature.

Figure 5. Gel profile of crude extracts of gut of DDVP-treated Rynchophorus phoenicis larva.

Notes: STD: Standard molecular mass, L1: Crude extract from control larva at 0 h and crude extract from larva L2–2 h, L3–4 h, L4–6 h, L5–8 h, L6–10 h, L7–12 h after treatment with DDVP.

Figure 6. Elution profiles of glutathione transferase from the gut of (A) control R. phoenicis larva and (B) DDVP-treated R. phoenicis larva using ion exchange chromatography.

Notes: Crude enzyme was applied to a DEAE-Sephadex A50 (1.5 × 25 cm) column previously equilibrated with 25 mM potassium phosphate buffer, pH 7.2 containing 1 mM EDTA and 1 mM 2-mercaptoethanol. The protein was eluted at a flow rate of 1 mL min⁻¹ and fractions of 5 mL each were collected. Fractions pooled: 33–37 (6B).

Figure 7. Elution profile of glutathione transferase on affinity chromatography-GSTrap 4B. Ion-exchange-purified enzyme was applied to a GSH-Sepharose 4B (1 × 1 cm) column previously equilibrated with PBS, pH 7.4.

Notes: The protein was eluted at a flow rate of 1 mL min⁻¹ and fractions of 1 mL each were collected. Bound protein was eluted with 10 mM GSH in 50 mM Tris–HCl buffer, pH 8.0. Fractions pooled: 13–18.

Table 1. Summary of purification of rplGSTc

Step	Total protein (mg)	Total activity (μmol min^−1)	Specific activity ( μmol min^−1 mg^−1)	Yield (%)	Fold
Crude GST	3,110.01 ± 194.2	8,011.22 ± 367.12	2.57 ± 0.16	100.00 ± 0.00	1.00 ± 0.00
Ion exchange chromatography	185.62 ± 11.5	2,164.25 ± 99.17	11.65 ± 0.72	27.10 ± 1.69	4.54 ± 0.28
GSTrap 4B	12.75 ± 2.34	665.05 ± 18.15	52.16 ± 5.28	8.32 ± 1.61	20.37 ± 2.43

Notes: Purification was conducted at 4°C. Values are mean ± SD of three repetitions.

Figure 8. Electrophorectograms of purified rplGSTc on (A) 10% polyacylamide gel slab at room temperature and (B) Native gel of 10% polyacrylamide.

Notes: Standard proteins ranging from molecular weight (MW) 17.27–103.14 kDa. Standard proteins ranging from molecular weight (MW) 29–272 kDa.

Figure 9. Double reciprocal plot of rplGSTc-catalyzed reaction. (A) Plots of 1/v_o vs. 1/[CDNB] at constant varied concentrations of GSH. (a) Secondary plot: plots of intercept or slopes in (A) vs. 1/[GSH]. (B) Plots of 1/v_o vs. 1/[GSH] at constant varied concentrations of CDNB. (b) Secondary plot: plots of intercepts or slopes in (B) vs. 1/[CDNB].