Characterization of the catalytic subunit of Trypanosoma cruzi cyclic AMP-dependent protein kinase.

The catalytic subunit of cyclic AMP-dependent protein kinase from Trypanosoma cruzi epimastigote forms was purified by ionic-exchange chromatography, affinity chromatography and sucrose gradient centrifugation. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, the purified preparations showed a main polypeptide band with a mobility of about 40 kDa. In Western blots this band immunoreacted with a polyclonal antibody specific for the catalytic subunit of bovine heart protein kinase A. Hydrodynamic and molecular parameters of this subunit are as follows: molecular weight, 40,000 +/- 3000; sedimentation constant, 2.8 +/- 0.3 S; Stokes' radius, 2.8 +/- 0.2 nm; frictional ratio, 1.28 +/- 0.05. Purified preparations of T. cruzi catalytic and regulatory subunits reconstitute a holoenzyme with a sedimentation constant. 8.6 +/- 1.17 S. This data together with those previously reported by Ulloa et al. [8] indicate that the T. cruzi cyclic AMP-dependent protein kinase holoenzyme is a tetramer with the structure R2C2 of about 200 kDa. The apparent Km of the catalytic subunit for ATP and histone IIA or kemptide as phosphate donor and acceptor, respectively, were 40 microM, 48.6 microM and 26 microM, respectively.