RAPID METHYLATION OF CELL-PROTEINS AND LIPIDS IN TRYPANOSOMA-BRUCEI

The fate of the [methyl-C-14] group of S-adenosylmethionine (AdoMet) i n bloodstream forms of Trypanosoma brucei brucei, was studied. Trypano somes were incubated with either [methyl-C-14]methionine, [U-C-14]meth ionine, S-[methyl-C-14]AdoMet or [S-35]methionine and incorporation in to the total TCA precipitable fractions was followed. Incorporation of label into protein through methylation was estimated by comparing mol ar incorporation of [methyl-C-14] and [U-C-14]methionine to [S-35]meth ionine. After 4-h incubation with [U-C-14]methionine, [methyl-C-14]met hionine or [S-35]methionine, cells incorporated label at mean rates of 2,880 pmol, 1,305 pmol and 296 pmol per mg total cellular protein, re spectively. Cells incubated with [U-C-14] or [methyl-C-14]methionine i n the presence of cycloheximide (50 mu g/ml) for four hours incorporat ed label eight- and twofold more rapidly, respectively, than cells inc ubated with [S-35]methionine and cycloheximide. [Methyl-C-14] and [U-C -14]methionine incorporation were > 85% decreased by co-incubation wit h unlabeled AdoMet (1 mM). The level of protein methylation remaining after 4-h treatment with cycloheximide was also inhibited with unlabel ed AdoMet. The acid precipitable label from [U-C-14]methionine incorpo ration was not appreciably hydrolyzed by DNAse or RNAse treatment but was 95% solubilized by proteinase K. [U-C-14]methionine incorporated i nto the TCA precipitable fraction was susceptible to alkaline berate t reatment, indicating that much of this label (55%) was incorporated as carboxymethyl groups. The rate of total lipid methylation was found t o be 1.5 times that of protein methylation by incubating cells with [U -C-14]methionine for six hours and differential extraction of the TCA lysate. These studies show T. b. brucei maintains rapid lipid and prot ein methylation, confirming previous studies demonstrating rapid conve rsion of methionine to AdoMet and subsequent production of post-methyl ation products of AdoMet in African trypanosomes.