Divalent cation hinder the solubilization of a tubulin kinase activity from Trypanosoma cruzi epimastigotes.
Trypanosoma cruzi epimastigotes were extracted under various conditions in order to examine the role of divalent cations in the solubilization of microtubule proteins. When epimastigotes were homogenized in the presence of 5 mM Mg+2 and 5 mM Ca+2, a protein kinase responsible for phosphorylating tubulin, as well as the tubulin that became phosphorylated, remained tightly associated with the parasite particulate and detergent-resistant fractions. On the contrary, tubulin kinase and its substrate were predominantly released into the parasite cytosolic and detergent-soluble fractions, when epimastigotes were extracted in the presence of 5 mM EDTA and 5 mM EGTA. These evidences demonstrated a divalent cation-dependent solubilization of the enzyme responsible for the phosphorylation of tubulin in T. cruzi epimastigotes and suggested a tight association between tubulin and this kinase. Under all conditions tested, tubulin kinase activity in epimastigote extracts was lower than the addition of the corresponding value in the parasite cytosolic and membranous fractions, suggesting the presence of a kinase inhibitor or regulatory subunit which also seemed to be modulated by divalent cations. Additionally, inhibition experiments in the presence of heparin, 2,3-bisphosphoglycerate and GTP established that the parasite tubulin kinase corresponded to a protein kinase CK2.
Uzcanga
Graciela
G
Departamento de Biología Celular, Universidad Simón Bolívar, Caracas, Venezuela.
Galán-Caridad
José Manuel
JM
Noris Suarez
Karem
K
Bubis
José
J
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D047390:Casein Kinase II; D004591:Electrophoresis, Polyacrylamide Gel; D008274:Magnesium / Q000494:pharmacology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects* / Q000378:metabolism; D012995:Solubility; D014349:Trypanosoma cruzi / Q000201:enzymology*; D014404:Tubulin / Q000378:metabolism*
D014404:Tubulin; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D008274:Magnesium; D002118:Calcium