Characterization of phosphorylation of a novel protein kinase in rice cells by capillary electrophoresis.

It was demonstrated that a separation of 20 amino acids constituting a protein and three phosphoamino acids that mostly frequently occur in eukaryotes was achieved within 15 min by capillary electrophoresis coupled with lamp-induced fluorescence detection. Fluorescein isothiocyanate was employed as the fluorescence label to facilitate the fluorescence detection of the 23 amino acid species. The fluorescent derivatization conditions and separation parameters including concentration of electrolyte, surfactant in buffer, applied voltage and sample injection were investigated in detail and optimized. The influence of buffer additives such as methanol, acetone and polyvinylpyrrolidone on separation selectivity and sensitivity were discussed. We showed that addition of 2% polyvinylpyrrolidone into the running buffer could dramatically enhance the separation selectivity of amino acids at the expense of a decrease of sensitivity of phosphoamino acids. Under the optimized conditions, the detection limits (S/N=2) ranged from 1.90 x 10(-8) M to 5.66 x 10(-8) M with an average efficiency of 620,000/m. The method was applied to characterization of the phosphorylation of a novel protein kinase RCaMBP (calcium/calmodulin-binding protein kinase) encoded by a cDNA newly isolated and cloned from rice. We verified that RCaMBP belonged to a type of Ser/Thr kinase, providing insight into its function in signal transduction.