Purification and sequencing of napin-like protein small and large chains from Momordica charantia and Ricinus communis seeds and determination of sites phosphorylated by plant Ca(2+)-dependent protein kinase.
The basic protein fraction from seeds of castor bean (Ricinus communis L.) contains 4732 Da and 4603 Da proteins phosphorylated in vitro by plant Ca(2+)-dependent protein kinase (CDPK). These proteins, RS1A and RS1B respectively, were purified by cation-exchange HPLC (SP5PW column) and reverse-phase HPLC (C18 column) and identified as napin-like protein small chains by Edman sequencing and electrospray ionization mass spectrometry (ESMS). The other R. communis 4 kDa small chains (RS2A, RS2B, RS2C and RS2D) are not phosphorylated by CDPK and neither is the corresponding 7332 Da large chain (RL) that forms 1:1 disulfide-linked complexes with RS2(A-D). RS1A/B is one of the best substrates found for plant CDPK (K(m) = 1.8 +/- 0.8 microM). RS2(A-D) (but not RL or RS1A/B) strongly inhibit calmodulin (CaM)-dependent myosin light chain protein kinase (MLCK) (IC50 = 0.25 microM) and inhibit the Ca(2+)-dependent enhancement of dansyl-CaM fluorescence. The basic protein fraction from seeds of bitter melon (Momordica charantia) also contains napin-like proteins that are 1:1 disulfide-linked complexes of a small chain (MS1, MS2, MS3 or MS4) and a large chain (ML). The M. charantia small chains were purified and completely sequenced by Edman degradation and ESMS. M. charantia small chains MS1, MS2, and MS4 (but not MS3) are phosphorylated by CDPK to unit stoichiometry on S21 within the sequence R17SCES21FLR. The R. communis small chain RS1A is phosphorylated on S34 within the sequence R31QSS34SRR. Both of these phosphorylation site motifs are consistent with those found for other plant CDPK substrates.
Neumann
G M
GM
School of Biochemistry, Trobe University, Bundoora, Vic, Australia.
Condron
R
R
Polya
G M
GM
D055516:2S Albumins, Plant; D000595:Amino Acid Sequence; D001665:Binding Sites; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002367:Ricinus communis / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D005638:Fruit / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D010947:Plants, Toxic; D011494:Protein Kinases / Q000378:metabolism*; D012639:Seeds / Q000737:chemistry; D017871:Calcium-Calmodulin-Dependent Protein Kinases
D055516:2S Albumins, Plant; D002147:Calmodulin; D010446:Peptide Fragments; D010940:Plant Proteins; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C050363:napin protein, Brassica napus