期刊
JOURNAL OF PROTEIN CHEMISTRY
卷 22, 期 6, 页码 591-599出版社
SPRINGER/PLENUM PUBLISHERS
DOI: 10.1023/B:JOPC.0000005509.60532.af
关键词
Trypanosoma cruzi; protein kinase; protein kinase CK1; protein phosphorylation/dephosphorylation; signal transduction
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 Angstrom. 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-aminoethyl)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.
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