Single-Molecule Analysis of Chemotactic Signaling in Dictyostelium Cells M. Ueda, Y. Sako, T. Tanaka, P. Devreotes, T. Yanagida

Supplementary Material

Preparation of Cy3-cAMP
cAMP (1.5 mmol) was dissolved in 18ml of 0.5 M sodium bicarbonate solution at pH 9.6 at 38 °C. 2.5 mmol of succinic anhydride was then added to this solution while stirring continuously. The solution was maintained at pH 9.6 by titration with 1N NaOH. The reaction was considered complete when the pH ceased to fall. After the pH was reduced to 3.0 with 1N HCl, the solution was left to allow the cAMP derivatives to precipitate. The precipitates were collected and washed in 100% EtOH by centrifugation, and then dried by evaporation. After reverse phase chromatography on Resource RPC (Pharmacia), the fractions containing 2'-O-succinyl cAMP were pooled and dried by evaporation. This method was originally developed to conjugate cAMP with N-methylisatoic anhydride at position 2'-O of ribose moiety (1). Preparation of 2'-O-succinyl cAMP was checked for its molecular weight, absorption maximum, and stability in 0.1M NaOH because succinylation of cAMP may give both 2'-O and N⁶ derivatives (2). The molecular weight of the derivative was revealed to be 429.3 by mass spectroscopy, which is expected for the 2'-O-succinyl cAMP (C₁₄H₁₆N₅O₉P) derivative. The absorption maximum of the derivative was 258 nm in water at pH 7.0, which is the same value as cyclic AMP itself, meaning that a succinyl group had not bound to the N⁶position of adenosine moiety (2). The cAMP derivative we prepared was unstable in 0.1M NaOH, indicating that it was the 2'-O derivative and not the N⁶ derivative (28).

The N-hyadroxysuccinimide ester of an orange fluorescent cyanine dye, Cy3 (Amersham) was incubated with ethylendiamine in 0.1M NaCO₃ at pH 9.3. The resulting ethylendiamine-Cy3 was purified by reverse phase chromatography. Cy3-cAMP was prepared by incubating 2'-O-succinyl cAMP (1 mg) with ethylendiamine-Cy3 (30 nmoles) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (1 mg) in 30 l of distilled water overnight at room temperature. Cy3-cAMP was purified by reverse phase chromatography and then by anion-exchange chromatography. The pooled fractions containing Cy3-cAMP were dried by evaporation, and then the product was dissolved in water and stored at -80 °C. The final product of Cy3-cAMP contained undetectable amounts of non-labeled cAMP. The molar ratio of Cy3 to cAMP was approximately 1 to 1 as determined from their extinction coefficients 14,650 M^-1cm^-1at 258 nm for cAMP, and 150,000 M^-1cm^-1 at 552 nm for Cy3.

References for supplementary information

1. T. Hiratsuka, J. Biol. Chem. 257, 13354 (1982).
2. J-G. Falbriard, T. H. Posternak, E. W. Sutherland, Biochim. Biophys. Acta 148, 99 (1967).