Analysis Of ～(35)S-CaM Binding To Arabidopsis Protoplasts

Our previous studies showed existence of apoplast CaM in the plant cell and CaM had many extracellular functions.So it supposed CaM may be one of important extracellular polypeptides and trigger the intracellular signal transduction by binding the receptor.In this study, radiolabelled ligand is used to investigate the binding characteristic of CaM and A. thaliana protoplasts.and chemical crosslinking is employed to explore binding proteins in the membrane.At first, (35)^S-CaM was produced using (35)^S-labeled amino acid mixture in E. coli..SDS-PAGE and autoradiograph indicated high-purified, high-specific radioactivity (35)^S-CaM was obtained.Electrophoresis of (35)^S-CaM is the same as that of unlabeled CaM with Ca²⁺ or EGTA; A quatitive of protoplasts was prepared by enzymolysis.Subsequently, the binding of (35)^S-CaM and A. thaliana protoplasts were processed, including association, dissociation, competitive and saturation analysis. The results show the binding of (35)^S-CaM and protoplasts is rapid, reversible, saturable and sensitive to proteinase; otherwise the specificity of binding was assessed with different competitors. K_d of 9. 2 nM and about 25,000 binding sites per protoplast were gained through saturation analysis.Eventually, two special bands were crosslinked in arabidopsis protoplast membrane by chemical crosslinker, and their molecular weights are about respectively 160kD and 60kD, which were receptor-like proteins.All these data indicate that there may be CaM binding sites in the arabidopsis protoplast and CaM binding has characteristic of receptor kinetics.