Fig. 11. Trapping and detection of a 68-kDa phosphoprotein formed by incubating Kdo₂-[4′-³²P]lipid IV_A with partially purified 4′-phosphatase/phosphotransferase.

A, a specific ³²P-labeled phosphoprotein, generated during a 5-min incubation of Kdo₂-[4′-³²P]lipid IV_A with enzyme (as described under “Experimental Procedures”) is resolved by SDS-polyacrylamide gel electrophoresis and detected by PhosphorImager analysis of the dried gel. Partially purified enzyme from either the Q-Sepharose step (lane 2, 6 μg of protein) or the heparin-agarose step (lanes 1 and 3; 2 μg of protein) was used. The enzyme for the reaction shown in lane 1 was first heated to 65 °C for 15 min. B, PtdIns reduces the level of the phosphoprotein generated during a 5-min incubation of Kdo₂-[4′-³²P]lipid IV_A with enzyme. Reactions were performed with identical amounts (2 μg) of heparin-agarose enzyme and Kdo₂-[4′-³²P]lipid IV_A in the absence (lane 1) or in the presence (lane 2) of PtdIns (0.5 mg/ml). After 5 min, the reactions were quenched with SDS and subjected to gel electrophoresis.