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.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
