FIG. 4.

Functional analysis of rPPX. (a) The PPX activity present in cell extracts from E. coli strain BL21(DE3)/pLysS transformed with pET-21b(+) carrying the c50_004-3 insert (SC21 cells) (gray bars) or in SC3 control cells carrying only the vector (white bars) was measured at the indicated temperatures as the decrease in the radioactivity of polyP used as a substrate. Incubation was done for 15 min. KU, kilounits (10³) of PPX activity. (b) TLC analysis of the reaction products obtained during the PPX assay. Samples (2 μl) from the reaction mixtures analyzed in panel a and a reaction mixture containing a cell extract from E. coli strain NR129 were taken at time zero and at 15 min of incubation and were applied to the origin of the plate. The arrows indicate the migration positions of [³³P]polyP₇₅₀ and H₃³²PO₄. Development of the TLC plates and autoradiography were done as described in Materials and Methods.