Subcellular distribution and partial characterization of the cyclic AMP-binding proteins of Trypanosoma brucei

We assayed the cyclic AMP-binding activity of Trypanosoma brucei by two well established methods, such as the one of Gilman (Proc. Natl. Acad. Sci. U.S.A. 67, 305-312, 1970) and Ueland & Doskeland (Biochem. J., 157, 117-126, 1976). The results indicate that the former technique underestimated the total amount of cyclic AMP bound by T. brucei homogenates by up to 7.5 fold. Similar results were obtained with other Trypanosomatidae such as Leishmania tropica, and Crithidia luciliae. The bulk of the cyclic AMP-binding proteins of T. brucei appeared soluble after centrifugation of post-large-granule extracts in isopycnic sucrose-gradients. Upon fractionation by DEAE-Sephacel column chromatography, two peaks of activity eluted which were responsible for all the specific cyclic AMP-binding activity present in the cytosol of T. brucei. These two activities, which we denominated as Peak 'a' and Peak 'b' respectively, differed in a number of properties such as sensitivity to proteases, stability to storage at -20 degrees C, displacement of cyclic AMP bound by adenine analogues, and coefficients of sedimentation.