A novel protein kinase from Brassica juncea stimulated by a protozoan calcium binding protein - Purification and partial characterization

A novel protein kinase (BjCCaBPk) from etiolated Brassica juncea seedlings has been purified and partially characterized. The purified enzyme migrated on SDS/PAGE as a single band with an apparent molecular mass of 43 kDa. Th e optimum pH for the kinase activity was 8.0. It was stimulated more than s ixfold by the protozoa Entamoeba histolytica calcium binding protein EhCaBP (10.5 nm) but not by calmodulin (CaM) when used at equimolar concentration . Moreover the kinase also did not bind CaM-Sepharose. There was neither in hibition of the kinase activity in the presence of W-7 (a CaM antagonist), KN-62 (a specific calcium/CaM kinase inhibitor) and anti-CaM Ig, nor any ef fect on BjCCaBPk activity of staurosporine (a protein kinase C inhibitor). Furthermore a CaM-kinase specific substrate, syntide-2, proved to be a poor substrate for the BjCCaBPk compared with histone III-S. The phosphorylatio n of histone III-S involved serine residues. Southern and Northern blot ana lysis showed the presence of EhCaBP homologues in Brassica. The data sugges t that BjCCaBPk may be a novel protein kinase with an affinity towards a ca lcium binding protein like EhCaBP.