Resolution of three Ca 2+-dependent protein kinases and endogenous substrate proteins from bitter gourd seeds

Abstract

A protein that is phosphorylated by plant Ca 2+-dependent protein kinase (CDPK) was isolated from seeds of bitter gourd ( Momordica charantia). This bitter gourd protein (referred to as BGP) was purified by a procedure involving batchwise elution from carboxymethylcellulose (CM-52), gel filtration, cation exchange HPLC and reversed phase HPLC. BGP preparations exhibit three bands (11 kDa, 7 kDa and 4 kDa, respectively) on SDS-PAGE, of which the 4-kDa material copurifies on SDS-PAGE with [ 32P]phosphoBGP phosphorylated by wheat CDPK or CDPKs isolated from M. charantia seeds. The 4-kDa [ 32P]phosphoBGP can be resolved from the other components after phosphorylation using [γ- 32P]ATP and CDPK and subsequent reversed phase HPLC. Electrospray ionization mass spectrometry (ESMS) of BGP revealed a major component with an average molecular mass of 11 450 Da, minor 11 287 Da and 11 563 Da components and other minor components corresponding to K + adducts of the major component. Reversed phase HPLC of BGP after treatment with 3 M guanidine HCl and 25% (v/v) 2-mercaptoethanol resolves three very similar small proteins (BGS1, BGS2 and BGS3) having average molecular masses of 3443 Da, 3606 Da and 3720 Da, respectively, and a larger protein (BGL) having an average molecular mass of 7850 Da. The 11 287-Da, 11 450-Da and 11 563-Da BGP components correspond within experimental error to 1:1 complexes of the large subunit (BGL) with a small subunit (BGS1, BGS2 or BGS3, respectively), with all complexes involving approximately three disulphide linkages (calculated masses 11 287 ± 2 Da, 11 450 ± 1 Da and 11 563 ± 2 Da, respectively). The 3606-Da BGS2 is a poor CDPK substrate in comparison with BGS1 and BGS3. Three CDPKs (CDPKs I, II and III) were resolved from M. charantia. All three CDPKs are absolutely dependent on millimolar Mg 2+ and about 10 −7–10 −6 M free Ca 2+ for maximal activity and phosphorylate BGP, histone III-S, bovine serum albumin, casein and myosin light-chain derived synthetic peptide (MLCP) (KKRAARATSNVFA-NH 2). CDPKs I and II phosphorylate the synthetic peptide kemptide (LRRASLG) better than CDPK III. CDPKs I, II and III are inhibited by poly- l-arginine (IC 50 values 52, 70 and 43 nM, respectively) and the calmodulin antagonist calmidazolium (IC 50 values about 30 μM).