Purification and sequencing of multiple forms of Brassica napus seed napin small chains that are calmodulin antagonists and substrates for plant calcium-dependent protein kinase.

Six napin small (S) subunits and six napin large (L) subunits were resolved from the seeds of kohlrabi (Brassica napus var. rapifera) by a procedure involving extraction, batchwise elution from carboxymethylcellulose (CM52) and reverse-phase HPLC after treatment with guanidine hydrochloride and 2-mercaptoethanol. The precise average molecular masses of the ca. 4.5 kDa small subunits and the ca. 10 kDa large subunits were determined by electrospray ionisation mass spectrometry (ESMS). The amino-acid sequences of six small subunits (S1A, S1B, S2, S3A, S3B and S4) were deduced from the ESMS-based masses of tryptic fragments, Edman sequencing and previously published data. The deduced structures were precisely consistent with this data and with the ESMS-based average molecular masses of these polypeptides. The structures of the small subunits (39-41 residues) are very similar with variations involving single substitutions at or near the N-terminus and 1 to 3 changes within the last 7 amino acids. Particular B. napus small and large chains are phosphorylated by plant Ca2+-dependent protein kinase (CDPK). The best site of phosphorylation on small chains is inferred to be either S34 or S39 of S1B. The napin-containing basic protein fraction from B. napus seeds largely abolishes the Ca2+-dependent fluorescence enhancement of dansyl-calmodulin and also inhibits calmodulin (CaM)-dependent myosin light chain kinase (MLCK). The resolved napin small chains also inhibit MLCK. All of the kohlrabi napin small chains, as well as homologous Brassicaceae small chains, have a central 23 amino-acid sequence that can potentially form an alpha-helix in which all the basic residues are located on one side. This structural element may be involved in the interaction of these proteins with CaM and the biological activity of antifungal proteins of this kind.