FIG. 4.

Tracer studies of incorporated [³H]cPD1. (A) The spheroplasts from 5 ml of culture of OG1X carrying pAM351 were incubated with 1.0 nM [³H]cPD1 at 37°C for 90 min and then lysed with dimethyl sulfoxide. Ninety-seven percent of the incorporated radioactivity was recovered in the lysed fraction. The lysate was subjected to reverse-phase HPLC and eluted with a linear gradient of 20 to 50% (30 min) acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 1.0 ml/min. The radioactive peak at 24 min comprises 88% of the incorporated radioactivity. The arrow indicates the retention time of cPD1. The dotted line indicates the concentration of acetonitrile. (B) The intact cells from 5 ml of culture of OG1X carrying pAM351 were incubated with 0.3 nM [³H]cPD1 at 37°C for 15 min, treated with lysozyme and mutanolysin, and then osmotically lysed. The cell extract was subjected to gel filtration HPLC. Fractions were collected at 30-s intervals. Tritium counts were measured in each fraction.