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Fig. 2. Binding of CaD39 and CaD39-AB to CaM. The binding of either CaD39 (filled circles) or CaD39-AB (open circles) to CaM was measured by a modified ELISA method (Lin et al., 1991). Wells in microtiter plate were coated with 100 µl of 5 µg ml^–1 bovine brain CaM at 4°C overnight. Bound Ca²⁺-CaM was incubated with increasing amounts (0-60 pmole) of CaD39 or CaD39-AB, subsequently with monoclonal antibody C21 against CaD, and then with horseradish-peroxidase-conjugated anti-mouse IgG. After a 30-minute incubation with substrate (ABTS), the color intensity (O.D.) was determined by an ELISA reader at 405 nm. Averages from three independent experiments were plotted in the figure, with error bars representing the standard deviation. Mutant CaD39-AB almost abolished its binding to Ca²⁺-CaM, whereas wild-type CaD39 binds to Ca²⁺-CaM in a saturable fashion.

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