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Figure 4. Enhancement of poly(P)ase activities following poly(P) treatment. (A) Detection of polyphosphatase activities by TLC analysis. Whole-cell extracts were prepared from cultured cells that had been treated with either poly(P), Na-PO4, or ß-GP + AA and untreated control cells. Poly(P)ase activities were detected by TLC analysis of reaction mixtures containing these cell extracts and with [32P]poly(P) as a substrate. Substrate [32P]poly(P) (long chain) remained at the origin of the TLC plate, and low-molecular-weight products, corresponding to short-chain poly(P) species, migrated to the top of the TLC plate following development by 1 M HCOOH and 2 M LiCl. These reactions were performed in triplicate and developed in three lanes. (B) Identification of low-molecular-weight labeled products by purified poly(P)ase (rPPX1) treatment. Poly(P)-treated cell extracts (day 11) were incubated with [32P]poly(P) under the same conditions as described in panel A and were further incubated with or without purified rPPX1 (2.2 x 103 units) (Wurst et al., 1995) for 1 hr at 37°C. The reaction products were also analyzed by TLC. (C) Identification of low-molecular-weight labeled products by longer-TLC plate. Poly(P)-treated cell extracts (day 16) were incubated with [32P]poly(P) under the same conditions as described in panel A and were analyzed by longer-TLC (20 cm). Lanes: 1, [32P]poly (P) hydrolyzed by 10 mM HCl for 5 min at 90°C; 2, [32P] orthophosphate; 3, degradation products of [32P]poly(P) treated with cell extract (day 16). Radioactive images of the TLC plates were visualized in a BAS2000 image analyzer (FUJIX, Tokyo, Japan).
