Figure 3.

Purified, native IQGAP1 contains copurifying, but substoichiometic calmodulin. (A) Native IQGAP1 was purified from bovine adrenal cytosol by FPLC using sequential cation exchange (S-Sepharose), gel filtration (Sephacryl S-300), and anion exchange (Q-Sepharose) columns. A Coomassie blue–stained gel of 5-μg samples of cytosol and each of the three column steps are shown in the upper part of the figure. The lower part of the figure illustrates corresponding Western blots for the 190-kD IQGAP1 subunit and calmodulin, as well as a calmodulin concentration series (8, 16, and 32 ng). Note that the 190-kD subunit was undetectable in cytosol at the exposure shown here, and that it became progressively enriched throughout the purification procedure. Note also that 5 μg of purified IQGAP1 contained ∼20 ng of calmodulin, or an ∼1:20 molar ratio of calmodulin to the 190-kD polypeptide. (B) To determine whether the calmodulin present in purified IQGAP1 represented a copurifying subunit of the native molecule or a trace contaminant, Q-Sepharose fractions that contained the 190-kD polypeptide were analyzed by Western blotting with anticalmodulin. Coelution of the 190-kD polypeptide and calmodulin were observed, although the calmodulin peak was more skewed than the 190-kD peak toward late-eluting fractions (see Discussion). Calmodulin thus appears to be a bona fide, albeit substoichiometric subunit of purified native IQGAP1.