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 prot ein 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 a mino acid species. The fluorescent derivatization conditions and separation parameters including concentration of electrolyte, surfactant in buffer, a pplied voltage and sample injection were investigated in detail and optimiz ed. The influence of buffer additives such as methanol, acetone and polyvin ylpyrrolidone on separation selectivity and sensitivity were discussed. We showed that addition of 2% polyvinylpyrrolidone into the running buffer cou ld dramatically enhance the separation selectivity of amino acids at the ex pense of a decrease of sensitivity of phosphoamino acids. Under the optimiz ed conditions, the detection Limits (S/N=2) ranged from 1.90(.)10(-8) M to 5.66(.)10(-8) M with an average efficiency of 620 000/m. The method was app lied to characterization of the phosphorylation of a novel protein kinase R CaMBP (calcium/calmodulin-binding protein kinase) encoded by a cDNA newly i solated and cloned from rice. We verified that RCaMBP belonged to a type of Ser/Thr kinase, providing insight into its function in signal transduction . (C) 2001 Elsevier Science B.V. All rights reserved.