EXTRACELLULAR CALMODULIN-BINDING PROTEINS IN PLANTS - PURIFICATION OFA 21-KDA CALMODULIN-BINDING PROTEIN

A 21-kDa calmodulin (CaM)-binding protein and a 19-kDa calmodulin-bind ing protein were detected in 0.1 M CaCl2 extracts of Angelica dahurica L. suspension-cultured cells and carrot (Daucus carota L.) suspension -cultured cells, respectively, using a biotinylated cauliflower CaM ge l-overlay technique in the presence of 1 mM Ca2+. No bands, or very we ak bands, were shown on sodium dodecyl sulfate-polyacrylamide gel elec trophoresis gels overlayed with biotinylated cauliflower CaM when 1 mM Ca2+ was replaced by 5 mM EGTA, indicating that the binding of these two CaM-binding proteins to CaM was dependent on Ca2+. Less 21-kDa CaM -binding protein was found in culture medium of Angelica dahurica susp ension cells; however, a 21-kDa protein was abundant in the cell wall. We believe that the 21-kDa CaM-binding protein is mainly in the cell wall of Angelica dahurica. Based on its reaction with periodic acid-Sc hiff (PAS) reagent, this 21-kDa protein would appear to be a glycoprot ein. The 21-kDa CaM-binding protein was purified by a procedure includ ing Sephadex G-100 gel filtration and CM-Sepharose cation-exchange col umn chromatography. The purity reached 91% according to gel scanning. The purified 21-kDa CaM-binding protein inhibited the activity of CaM- dependent NAD kinase and the degree of inhibition increased with augme ntation of the 21-kDa protein, which appeared to be the typical charac teristic of CaM-binding protein.