FIG. 1.

In vitro MalQ activity with maltose as substrate. (A) The reaction was performed with 4.4 μg of C-terminally His₆-tagged MalQ in a total volume of 16 μl containing 250 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, and different concentrations of unlabeled glucose. After 10 min of incubation at room temperature, [¹⁴C]maltose (final concentration, 75 μM) was added to the mixture, followed by incubation for another 20 min at 30°C. Portions (5 μl) of the reaction were spotted. The figure represents the autoradiogram of the TLC plate. The standards were as follows: lane 1, [¹⁴C]glucose; lane 2, [¹⁴C]maltose; lane 3, [¹⁴C]maltotriose. Glucose additions to the enzymatic assays were as follows: lane 4, no glucose; lane 5, 0.0002 mM; lane 6, 0.001 mM; lane 7, 0.002 mM; lane 8, 0.01 mM; lane 9, 0.02 mM; lane 10, 0.1 mM; lane 11, 0.2 mM; lane 12, 1.0 mM; lane 13, 2.0 mM; lane 14, 10 mM. (B) The reaction was performed as in panel A with the additional 50 mM MgCl₂, 25 mM ATP, and 100 μM glucose without (lane 3 to 7) and with 1.5 μg of Glk (lanes 8 to 12). After 0, 1, 2, 5, and 20 min, 4 μl of the reaction was spotted. The standards were as follows: lane 1, [¹⁴C]glucose; lane 2, [¹⁴C]maltose; lane 13, [¹⁴C]glucose-6-phosphate.