Figure 3.

B. subtilis pxpA, pxpB, and pxpC deletant cells accumulated OP and lack 5-oxoprolinase activity. A, the amount of OP accumulated in cells and medium of 3-ml cultures grown with ammonium as nitrogen source to an optical density (600 nm) of 1.43 ± 0.02 (4.3 ± 0.3 × 10⁸ cells). Data are mean ± S.E. of six independent cultures. B, OPase activity in the desalted lysates of wild-type cells grown with OP as the nitrogen source or wild-type and deletant cells grown with ammonium as the nitrogen source. Assays (50 μl) contained 50 mm Tris-HCl, pH 8.0, 10 mm MgCl₂, 5 mm ATP (unless indicated otherwise), 2 mm glutamate, and 30 μm OP (0.1 μCi [³H]OP) and were started by adding 25 μl of lysate (10–17 mg of protein ml⁻¹). Assays were incubated at 37 °C for 15 min; glutamate was isolated by Dowex-50 (H⁺) chromatography and analyzed by scintillation counting. Data are the mean ± S.E. of three independent lysate preparations. †, not detectable (detection limits: 0.01 μg of OP in cells; 0.09 μg of OP in medium; 0.02 pmol min⁻¹ mg⁻¹ protein in assays).