Three basic proteins, M1, M2A and M2B, that are substrates for plant Ca(2+)-dependent protein kinase (CDPK) were purified from seeds of yellow mustard (Sinapis alba L.) by a protocol involving batchwise chromatography on carboxymethylcellulose (CM52), cation-exchange HPLC on an SP5PW column and reversed-phase HPLC on a C18 column. The complete amino-acid sequences of these proteins have been determined employing Edman sequencing and electrospray ionization mass spectrometry (ESMS) applied to the proteins and their tryptic and chymotryptic fragments. M1 (observed mass 5676.8 +/- 1.0 Da; calculated mass 5677.57 Da), M2A (observed mass 5704.8 +/- 0.8 Da; calculated mass 5704.60 Da) and M2B (observed mass 5839.5 +/- 1.2 Da; calculated mass 5838.78 Da) have been identified as gamma-thionins, which are potent antifungal proteins. M1, M2A and M2B are phosphorylated by plant CDPK on Ser residues, the site of phosphorylation on M2A being S8 as directly confirmed by Edman sequencing and mass spectrometry of the chymotryptically generated phosphopeptide CQRPS(HPO3)GTW11. M1 and M2A have apparent calmodulin (CaM) antagonist activity with IC50 values of 4.8 +/- 1.3 microM and 5.5 +/- 1.5 microM, respectively, for inhibition of CaM-dependent myosin light chain kinase (MLCK). M2A and/or M2B interacts with dansyl-CaM in both the presence and absence of calcium.