Figure 2.

Asp1^371–920 specifically hydrolyzes the 1-diester phosphate of 1-InsP₇ and 1,5-InsP₈. Panel A: Asp1^371–920 (0.7 μg/mL) was incubated in phosphatase-assay buffer (see Materials and Methods) with 10 μM of the indicated test substrate (either InsP₆ or the indicated PP-InsP). Reactions were assayed for phosphate release. Panel B: Phosphate release from 1-InsP₇ (assayed as described for Panel A) was determined following incubations with either10 μg/mL Asp1^371–920 for 30 min (left bar), or 10 μg/mL DIPP1 for 30 min (middle bar), or Asp1^371–920 for 30 min followed by DIPP1 for 30 min (right bar). Panel C: Phosphate release was measured in the same conditions described for Panel B, except 1,5-InsP₈ replaced 1-InsP₇ as substrate. For both panels B and C, the reactions catalyzed by DIPP1 and Asp1^371–920 are described above the bar graphs. Panel D, E, and F depict HPLC analysis of incubations (see Materials and Methods) in which trace amounts of 1-[³H]InsP₇ or 5-[³H]InsP₇ or 1,5-[³H]InsP₈, respectively, were each incubated with Asp1^371–920 (0.041 μg/mL) for either 0 min (circles) or 30 min (squares) in the phosphatase-assay buffer. The chromatographic peaks are labeled with the nature of the material and their elution peak (fraction number). HPLC data are representative of three experiments. Phosphate release data are means ± S.E., n ≥ 3.