Fig. 1.   HPLC analysis of the methylation reaction products. The products of the enzymatic reactions were analyzed by HPLC as described under "Experimental Procedures." The retention times of the standards (sarcosine (S), dimethylglycine (D), and betaine (B)) are shown by triangles on the x axis. A, synthesis of methylation products in a reaction mixture containing A. halophila cell extract. 25 mM glycine (white circles), 25 mM sarcosine (black square), or 25 mM dimethylglycine (black circle) and 4 mM S-adenosyl-L-[methyl-¹⁴C]methionine were used as substrates. All methylation intermediates from glycine to betaine are detected. The relative amount of reaction products depends on the substrate used. Identical results were obtained with E. halochloris cell extract (data not shown). B, synthesis of methylation products in a reaction mixture containing 0.53 mg/ml purified E. halochloris GSMT and 0.7 mg/ml purified A. halophila SDMT. 1.25 mM glycine (white circle) or 20 mM glycine (black square) and 8 mM S-adenosyl-L-[methyl-¹⁴C]methionine were used as substrates. The relative amount of products is dependent on the concentration of glycine in the reaction mixture.