FIGURE 11.

Enhanced binding of PFK-1 to calmodulin-agarose mediated by palmitoyl-CoA or S-hexadecyl-CoA. PFK-1 (1 μm) was incubated in the presence or absence of palmitoyl-CoA (10 μm) or S-hexadecyl-CoA (10 μm) for 1 h at 35 °C prior to application to a column of calmodulin-agarose (0.6 ml) equilibrated with buffer in the presence of 1 mm CaCl₂. Following washing of the column with three 1-ml volumes (W1–W3) of equilibration buffer, bound PFK-1 was eluted with buffer containing EGTA. Samples (50 μl) of each fraction were subjected to SDS-PAGE and silver staining to visualize the amount of PFK-1 in each fraction by densitometry as described under “Experimental Procedures.” Results are representative of four separate experiments. Abbreviations used are as follows: L, load fraction; F, flow-through fraction; W, wash fraction; E, elution fraction.