Abstract
A modification of the adenylyl cyclase assay method of Y. Salomon, C. Londos, and M. Rodbell (1974, Anal. Biochem. 48 , 541–548) is described. It makes the method applicable to the determination of guanylyl cyclase in subcellular tissue fractions. The modification consists of performing the Dowex 50 chromatography at acid pH in a column that has bed volume that is three times as large. This modification results in low blanks and allows for reuse of both Dowex 50 and aluminum oxide columns. A modification of the method of Symons [1974, Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 29, pp. 105–115, Academic Press, New York] for synthesis of [α- 32 P]nucleoside triphosphates is also presented. It allows all steps to be carried out within 5 h in a single reaction vessel. Synthesized NTPs are then purified by DEAE-Sephadex A-25 (HCO 3 − form) using a linear ammonium bicarbonate gradient. Its applicability to synthesis of [α- 32 P]GTP, [α- 32 P]dGTP, and [α- 32 P]ATP having specific activities of up to 75 Ci/mmol is shown.
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