Purification and Sequencing of Radish Seed Calmodulin Antagonists Phosphorylated by Calcium-Dependent Protein Kinase

Abstract

A family of radish (Raphanus sativus) calmodulin antagonists (RCAs) was purified from seeds by extraction, centrifugation, batch-wise elution from carboxymethyl-cellulose, and high performance liquid chromatography (HPLC) on an SP5PW cationexchange column. l h i s RCA fraction was further resolved into three calmodulin antagonist polypeptides (RCA1, RCAZ, and RCA3) by denaturation i n the presence of guanidinium HCI and mercaptoethanol and subsequent reverse-phase HPLC on a Ca column eluted with an acetonitrile gradient in the presence of 0.1% trifluoroacetic acid. The RCA preparation, RCA1, RCA2, RCA3, and other radish seed proteins are phosphorylated by wheat embryo Caz+-dependent protein kinase (CDPK). The RCA preparation contains other CDPK substrates in addition to RCAl, RCAZ, and RCA3. The RCA preparation, RCAl, RCA2, and RCA3 inhibit chicken gizzard calmodulin-dependent myosin light chain kinase assayed with a myosin-light chain-based synthetic peptide substrate (fifty percent inhibitory concentrations of RCA2 and RCA3 are about 7 and 2 ~LM, respectively). N-terminal sequencing by sequential Edman degradation of RCAl, RCA2, and RCA3 revealed sequences having a high homology with the small subunit of the storage protein napin from Brassica napus and with related proteins. l h e deduced amino acid sequences of RCA1, RCA2, RCA3, and RCA3' (a subform of RCA3) have agreement with average molecular masses from electrospray mass spectrometry of 4537, 4543, 4532, and 4560 kD, respectively. The only sites for serine phosphorylation are near or at the C termini and hence adjacent t o the sites of proteolytic precursor cleavage.

The 58-Kilodalton Calmodulin-Binding Glutamate Decarboxylase Is a Ubiquitous Protein in Petunia Organs and Its Expression Is Developmentally Regulated

“…With respect to the possible function of CaM in seeds during germination, it is interesting that recently a number of small proteins purified from radish seeds were identified as potent CaM antagonists (Polya et al, 1993). Moreover, a significant decrease in these antagonists and a simultaneous increase in CaM levels were observed during early phases of germination, implying that CaM-regulated processes are activated (Cocucci and Negrini, 1988).…”

Extracellular localization of napin in the embryogenic tissues of Brassica napus spp. oleifera

“…It has been shown that napin has a calmodulin antagonist activity and it has the potential calmodulininteracting structural elements (Polya et al 1993;Neumann et al 1996b, c). Oxidised napin complexes can be effective proteinase inhibitors and it was suggested that this might contribute to the biological activity of this protein as an anti-fungal agent (Neumann et al 1996a, b;Polya 1997).…”

Napins, 2S albumins, are major allergens in oilseed rape and turnip rape

“…24,25 In vitro bioactivities, like enzyme inhibition and antifungal activity of 2S albumins, have been reported, but the in vivo significance of these activities is unknown. 26,27 2S albumins have been shown to represent allergens, for example, in sunflower seed, white mustard, oriental mustard, sesame seed, brazil nut, and castor bean. 19,20,[28][29][30][31] At the moment, the route of sensitization of oilseed rape or turnip rape allergy is unclear.…”