PLANT PHYSIOLOGY , Vol 101, Issue 2 545-551, Copyright © 1993 by American Society of Plant Biologists
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CELL BIOLOGY AND SIGNAL TRANSDUCTION |
Purification and Sequencing of Radish Seed Calmodulin Antagonists Phosphorylated by Calcium-Dependent Protein Kinase
G. M. Polya, S. Chandra and R. Condron
Department of Biochemistry, La Trobe University, Bundoora, Victoria, 3083, Australia
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 cation-exchange column. This RCA fraction was further resolved
into three calmodulin antagonist polypeptides (RCA1, RCA2, and RCA3) by
denaturation in the presence of guanidinium HCI and mercaptoethanol and
subsequent reverse-phase HPLC on a C8 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 Ca2+-dependent protein kinase (CDPK). The RCA preparation
contains other CDPK substrates in addition to RCA1, RCA2, and RCA3. The RCA
preparation, RCA1, 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 [mu]M, respectively).
N-terminal sequencing by sequential Edman degradation of RCA1, 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.
The deduced amino acid sequences of RCA1, RCA2, RCA3, and RCA3[prime] (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 to the sites of proteolytic precursor
cleavage.
