Crithidia luciliae: Effect of Purine Starvation on S-Adenosyl-L-Methionine Uptake and Protein Methylation

Abstract

The utilization of S-adenosyl-L-[ methyl- 3H]methionine ([ 3H- methyl]AdoMet) by Crithidia luciliae was assessed under nutrient-replete and purine-starvation conditions. Uptake experiments with intact cells demonstrated that the radiolabel from this molecule was accumulated by purine-starved organisms at a rate ∼10-fold greater than that observed in those cultivated in nutrient-replete medium. Purine-starved cells also incorporated the radiolabel into trichloroacetic acid insoluble material at an ∼10-fold faster rate than nutrient-replete cells. No differences, however, were observed in the intracellular levels of AdoMet and its metabolites between organisms cultivated under the two conditions. Results of comparative labeling studies with [ 3H- methyl]AdoMet, S-adenosyl-L-[ carboxyl- 14C]methionine, L-[ methyl- 3H] methionine and L-[ 35S]methionine in the presence and absence of cycloheximide demonstrated that the incorporation of label from [ 3H- methyl]AdoMet was due to transmethylation and was independent of protein synthesis. Further, ∼15 methylated protein bands were identified by SDS-PAGE analysis. Lysates from both purine-starved and nutrient-replete organisms demonstrated similar levels of activity of three protein methyltransferases (PMI, II, III). The differences observed in [ 3H- methyl]AdoMet utilization between purine-starved and nutrient-replete C. luciliae may reflect the enhanced purine transport capacity which results from purine starvation.