PURIFICATION AND SEQUENCING OF MULTIPLE FORMS OF BRASSICA-NAPUS SEED NAPIN LARGE CHAINS THAT ARE CALMODULIN ANTAGONISTS AND SUBSTRATES FOR PLANT CALCIUM-DEPENDENT PROTEIN-KINASE

Six napin large (L) chains (as well as six napin small chains) were re solved from the seeds of kohlrabi (Brassica napus var. rapifera) by a procedure involving extraction, batchwise elution from carboxymethylce llulose (CM52) and reversed-phase HPLC after treatment with guanidine hydrochloride and 2-mercaptoethanol. The precise average molecular mas ses of the circa 4.5 kDa small subunits and the circa 10 kDa large sub units were determined by electrospray ionisation mass spectrometry (ES MS). Of six large subunits resolved (L1A, LIE, L1C, L2A, L2B and L2C), the complete amino acid sequences of four (LIA, L2A, L2B and L2C) and the near-complete sequences of two (LIE and L1C) were deduced from th e ESMS-based masses of tryptic fragments, Edman sequencing and previou sly published data. The deduced structures are precisely consistent wi th this data and with the ESMS-based average molecular masses of these polypeptides. ESMS analysis of unreduced napin extract revealed only seven circa 14.5 kDa complexes, the observed masses being in close agr eement with those calculated for 1:1 complexes of particular small and large subunits assuming four disulfides in each napin complex. The st ructures of the napin large subunits (86-91 residues) are very similar and all amino acid differences observed are confined to only 25 posit ions. The L2A, L2B and L2C large chains (but not the L1A, L1B and L1C large chains) are phosphorylated well by plant Ca2+-dependent protein kinase (CDPK). The CDPK-catalyzed phosphorylation site on the large ch ain L2A is inferred to be S-57 within the sequence LQQVIS(57)RIYQT (th e site being S-60 within the same sequence in L2B and L2C). The napin- containing basic protein fraction from B. napus seeds largely abolishe s the Ca2+-dependent fluorescence enhancement of dansyl-calmodulin and also inhibits calmodulin (CaM)-dependent myosin light chain kinase (M LCK). The resolved napin long chains also inhibit MLCK. Each kohlrabi large chain contains 2 sequences (corresponding to L(10)-Q(20) and Q(5 1)-L(64) Of L1A) which have the potential to form amphipathic a-helice s. Each large chain also contains a Q-rich 19 amino acid sequence (cor responding to L(30)-Q(48) Of LIA) which has the potential to form a '2 -sided' alpha-helix with basic residues confined to one side. These st ructural elements may be involved in the inferred interaction of these proteins with CaM and may be relevant to the biological activity of a ntifungal proteins of this kind.