FIG 4.

ChBD-mediated binding of SptC to chitin. (A) Chitin-binding capacities of SptCS230A, the intermediate forms (I^WT, I^ΔChBD, I^ΔPkdD, and I^ΔCTE), the mature form (M^WT), and the C-terminal fragments (ChBD*, PkdD*, and CTE*). Each protein (5 μg/ml) was incubated in 300 μl of buffer A (50 mM Tris-HCl, 10 mM CaCl₂ [pH 8.0]) containing 5 M NaCl at 0°C for 30 min in the absence (lane 1) or presence (lane 2) of 5 mg of insoluble chitin. After centrifugation, 120 μl of the supernatant was subjected to SDS-PAGE analysis. Numbers given on the right of the images indicate the densitometric ratios of unbound protein (lane 2) to the control (lane 1) and are expressed as the means ± SDs of three independent experiments. (B) Salt dependence of the binding of the CTE* to chitin. CTE* (5 μg/ml) was incubated at 0 or 37°C for 30 min without chitin and NaCl (lane C) or with 5 mg of insoluble chitin in 300 μl of buffer A containing different concentrations of NaCl (1 to 5 M). After centrifugation, the supernatants were subjected to SDS-PAGE analysis.