title: The cAMP receptor protein of Trypanosoma cruzi.
abstract: The widest used method to determine cAMP-binding activity in cell-free extracts (Gilman, A. G. (1970) Proc. Natl. Acad. Sci. U.S.A. 67, 305-312) underestimated by about 10-fold the total amount of [3H]cAMP bound by crude extracts of cultured forms of Trypanosoma cruzi (epimastigotes), when compared with the results obtained by the modified Millipore filter technique described by Doskeland and Ueland (Doskeland, S. O., and Ueland, P.M. (1977) Biochem. J. 165, 561-573). After column chromatography on DEAE-Sephacel, the cAMP-binding activity eluted as a single symmetrical peak at about 210 mM NaCl, totally separated from two peaks of cAMP-independent phosphotransferase activities which eluted, respectively, at 90 and 270 mM NaCl. These two protein kinases showed similar specificities for exogenous substrates, preferring in this order: protamine greater than casein greater than histone H2b. Photoaffinity labeling of cell-free extracts with 8-azido[32P]adenosine 3':5'-monophosphate and autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis specifically labeled only one protein of Mr = 62,000. From these results it is concluded that T. cruzi epimastigotes possess one single cAMP-binding protein of monomeric Mr = 62,000, not associated with a phosphotransferase activity and probably very different in nature to known regulatory subunits of protein kinases, given the results obtained with the Millipore filtration technique.
mesh_terms: D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001386:Azides; D002417:Cattle; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D009206:Myocardium / Q000378:metabolism; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011953:Receptors, Cyclic AMP / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000378:metabolism*
chemical_list: D000345:Affinity Labels; D001386:Azides; D011953:Receptors, Cyclic AMP; C017042:8-azidoadenosine-3',5'-monophosphate; D000242:Cyclic AMP; D011494:Protein Kinases