Time course of polyP-dependent AMP phosphorylation. The reaction mixtures (100 μl) containing 50 mM Tris⋅HCl (pH 8.0), 50 mM ammonium sulfate, 10 mM MgCl₂, 5 mM AMP, polyP (75 mM of phosphate, chain length, 15–20), 4 μg purified PPK, and a cell lysate (0.8 μg of proteins) was incubated at 30°C. The ADP and ATP formed were monitored and plotted as the sum of both levels. ■, No lysate; ⧫, the cell lysate of JM109 harboring the control vector pTrc99A; ●, the cell lysate of JM109 overproducing ADK.

Autoradiogram showing phosphotransfer from [³²P]polyP to AMP by the combined action of both purified enzymes. Each or both of 4 μg purified PPK and ADK were used for enzyme samples in the PAP assay with [³²P]polyP. Each sample was analyzed by PEI-TLC followed by autoradiography: lanes 1 and 6, reaction compounds with no enzyme; lanes 2 and 7, reaction compounds containing PPK; lanes 3 and 8, reaction compounds containing ADK; lanes 4, 5, and 9, reaction compounds containing PPK and ADK; lanes 1–4, AMP was omitted from the reaction mixtures. Other details are given in Materials and Methods.

A stoichiometry of PPK and ADK required for the PAP activity. The purified PPK (25 pmol) and various doses of the purified ADK (1.3, 2.5, 6.3, 13, 25, and 63 pmol) were used for enzyme samples in the PAP assay. The relative activity to the value observed for 25 pmol of both enzymes was plotted against the dose of ADK added, the value of which was expressed relative to the amount of PPK.

Complex formation between PPK and ADK in the presence of polyP. The reaction mixture (1 ml) containing 0.5 mg of each or both of PPK and ADK, 50 mM Tris⋅HCl (pH 8.0), 50 mM ammonium sulfate, 10 mM MgCl₂, 5 mM AMP and polyP (375 mM in terms of phosphate, chain lengths, 15–20) was incubated at 30°C for 30 min. The resultant mixtures were applied onto AKTA FPLC column system with HiLoad 16/60 Superdex 200pg (Amersham Pharmacia) and separated by isocratic elution with polyP-containing buffer [50 mM Tris⋅HCl, pH 8.0, 50 mM ammonium sulfate, 10 mM MgCl₂ and polyP (15 mM in terms of phosphate, chain lengths, 15–20)]. After the void-volume (45 ml) of the eluent was wasted, each 5 ml of the eluent was fractionated and defined as fractions 1–10, respectively. Fraction 1 is the largest molecular weight fraction in this chromatography. (A) The results of SDS/PAGE analysis of fractions 1–10. Proteins in each fractions (200 μl) were precipitated by 5% trichloroacetic acid and analyzed. The fractions in the case that only ADK was incubated followed by the chromatography were analyzed in Aa, those in the case that only PPK was in Ab, and those in the case that PPK and ADK together were in Ac. Corresponding fraction numbers analyzed were designated at the top of each lane. (B) The result of polyP-forming assay of the fractions 1–10. The fractions in the case that only PPK was incubated followed by the chromatography were analyzed in Bb′, those in the case that PPK and ADK together were in Bc′. Corresponding fraction numbers analyzed were designated at the top of each lane.