Figure 3.

Purified Arg1 specifically phosphorylates IP₃ to produce IP₄ and IP₅. (A) Recombinant Arg1 (49 kDa, including the His₆  tag) was purified by TALON cobalt affinity chromatography followed by ion exchange chromatography (IEC). (B) Purified Arg1 (1–10 ng/µl reaction, as indicated) was incubated with ATP and IP₃ and the reaction products analyzed by polyacrylamide gel electrophoresis and Toluidine Blue staining. Bands representing different IP species and ATP are indicated. (C) Kinetics of Arg1 activity. For these assays, purified Arg1 (20 ng) was incubated with ATP (50–500 µM) and 200 µM of IP₃ in 10 µL reactions at room temperature for 0–30 min. The consumption of ATP was monitored using bioluminescence. The assay was performed twice, each time in triplicate. V: reaction velocity.