Protein kinase CK1 from Trypanosoma cruzi.
A protein kinase activity, which uses casein as a substrate, has been purified to homogeneity from the epimastigote stage of Trypanosoma cruzi, by sequential chromatography on Q sepharose, heparin sepharose, phenyl sepharose, and alpha-casein agarose. An apparent molecular weight of 36,000 was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration chromatography and sedimentation analyses demonstrated that the purified native enzyme is a monomer with a sedimentation coefficient of 2.9 S. The hydrodynamic parameters indicated that the shape of the protein is globular with a frictional ratio f/f(o) = 1.36 and a Stokes radius of 27.7 A. When two selective peptide substrates for protein kinases CK1 and CK2 were used (RRKDLHDDEEDEAM. SITA and RRRADDSDDDDD, respectively), the purified kinase was shown to predominantly phosphorylate the CK1-specific peptide. Additionally, the enzyme was inhibited by N-(2-amino-ethyl)-5-chloroisoquinoline-8-sulfonamide, a specific inactivator of CK1s from mammals. Based on these results, we concluded that the purified kinase corresponds to a parasite CK1.
Calabokis
Maritza
M
Departamento de Biología Celular, Universidad Simón Bolívar, Apartado 89.000, Valle de Sartenejas, Caracas 1081-A, Venezuela.
Kurz
Liliana
L
Gonzatti
Mary I
MI
Bubis
José
J
D000595:Amino Acid Sequence; D000818:Animals; D047388:Casein Kinases; D002850:Chromatography, Gel; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000201:enzymology*
D007546:Isoquinolines; D047428:Protein Kinase Inhibitors; C058815:N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide; D011494:Protein Kinases; D047388:Casein Kinases