Calcineurin immunoprecipitated from bovine brain extract contains no detectable Ni2+ or Mn2+.
Purified calcineurin phosphatase is converted upon incubation in millimolar Ni2+ or Mn2+ to an active form by association with these metal activators. The bound metal ion is not dissociable from calcineurin by dialysis or gel filtration, but can be released upon prolonged incubation of the enzyme with Ca2+/calmodulin or chelating agents (Pallen, C.J., and Wang, J.H. (1986) J. Biol. Chem. 261, 16115-16120). The present study has been undertaken to test the possibility that calcineurin in brain may contain tightly bound Ni2+ or Mn2+. A monoclonal antibody (VA1) immunoaffinity matrix was prepared and shown to affect specific precipitation of calcineurin from crude bovine brain extract. Using [3H]-, [63Ni2+]-, and [54Mn2+]calcineurin added to the extract as radioactive tracer, it was found that up to 80% of the calcineurin could be immunoprecipitated, and that more than 50% of the originally bound metal ions could be detected in the immunoprecipitate. When samples of calcineurin immunoprecipitated from brain extracts were analyzed by atomic absorption spectroscopy, Ni2+ and Mn2+ were not detected, whereas, Zn2+, a constitutive metal of calcineurin (King, M. M., and Huang, C. Y. (1984) J. Biol. Chem. 259, 8847-8856) was found in the expected amount. The result suggests that calcineurin in brain does not contain tightly associated Ni2+ or Mn2+.
Rao
J
J
Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
Wang
J H
JH
D000818:Animals; D000906:Antibodies; D000936:Antigen-Antibody Complex / Q000032:analysis; D001921:Brain / Q000201:enzymology*; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000302:isolation & purification*; D002417:Cattle; D008345:Manganese / Q000032:analysis*; D009532:Nickel / Q000032:analysis*; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification*; D011485:Protein Binding
D000906:Antibodies; D000936:Antigen-Antibody Complex; D002148:Calmodulin-Binding Proteins; D008345:Manganese; D009532:Nickel; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases