Purification and sequencing of yellow mustard seed napin small and large chains that are phosphorylated by plant calcium-dependent protein kinase and are calmodulin antagonists

Abstract

A multiplicity of small (S) and large (L) napin subunits were purified from yellow mustard ( Sinapis alba L.) seeds by a protocol involving extraction, successive batch-wise cation exchange chromatography on carboxymethylcellulose (CM52), cation exchange HPLC on an SP5PW column and reversed phase HPLC on a C18 column. Initial cation exchange HPLC resolved 4 major zones of proteins (M1, M2, N1 and N2) that can be phosphorylated by plant Ca 2+-dependent protein kinase (CDPK). Electrospray ionization mass spectrometry (ESMS) revealed that M1 and M2 are 6 kDa proteins, later identified as γ-thionin-related proteins. ESMS of fractions N1 and N2 revealed the presence of 14.5 kDa proteins identified as napin complexes, each composed of a single small subunit linked to a single large subunit and involving 4 disulphide linkages. The napin complexes (N1A, N1B, N1C, N1D, N2A, N2B and N2C) were disrupted and the constituent small subunits (S1, S2 and S3) and large subunits (L1A, L1B, L1C, L2A, L2B and L2C) were resolved by reversed phase HPLC and precise average molecular masses determined by ESMS. The small and large subunits have average molecular masses of about 4.4 kDa and 10.1 kDa, respectively. The masses of each napin complex can be precisely accounted for from the masses of the constituent subunits. Thus the major complex N2A (14 569 ± 3 Da) is evidently composed of S3 (4434.0 ± 1.5 Da) and L2A (10 142.5 ± 1.5 Da) and involves 4 disulphides (loss of 8.0 Da), the expected mass of S3 + L2A-8H being 14 569 ± 2 Da. The yellow mustard napin large chain L2A is phosphorylated by wheat CDPK on Ser 60 within the sequence LQHVIS 60RIY. The complete sequence of this and other large (and small) napin subunits were determined from Edman sequencing and/or ESMS data by comparison with published napin sequences. Yellow mustard seed CM52-binding fractions decrease the Ca 2+-dependent fluorescence emission of dansyl-CaM and yellow mustard small and large chains inhibit CaM-dependent myosin light chain kinase (MLCK).