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Fig. 5 | BMC Plant Biology

Fig. 5

From: Fruit ripening-associated leucylaminopeptidase with cysteinylglycine dipeptidase activity from durian suggests its involvement in glutathione recycling

Fig. 5

SDS-PAGE and western blot (a) metal ion dependency (b) and optimal pH (c) of recombinant DzLAP1. Lane M: standard protein ladder. Lanes 1 and 3: crude rDzLAP1. Lanes 2 and 4: purified rDzLAP1. Two micrograms crude and purified proteins were loaded onto SDS gel (a). Three and one-half micrograms rDzLAP1 was incubated with 7.5 mM Cys-Gly in 25 mM K3PO4 buffer (pH 7.2) in the presence of 0 mM and 1 mM Ca2+, Zn2+, Mg2+, Ni2+, and Mn2+ at 37 °C for 15 min. The total reaction volume was 50 μL. Enzyme activity was measured spectrophotometrically by a modified acidic ninhydrin method [39] at 560 nm (A560) (b). The optimum pH for rDzLAP1 was established by incubating 3.5 μg enzyme with 7.5 mM Cys-Gly and 1 mM Mg2+ at various pH (acetate buffer, pH 4–6; phosphate buffer, pH 6–8; Tris-HCl buffer, pH 8–9.5; glycine-NaOH buffer, pH 9.5–11) at 37 °C for 15 min (c). Enzyme activity was calculated relative to a maximum of 100%

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