The influence of choline on the utilization of methionine for protein synthesis by primary cultures of chick hepatocytes

Abstract

Two experiments were performed in order to determine the methionine and choline requirements for maximum protein synthesis by primary cultures of chick hepatocytes. In experiment 1, a 4 x 8 factorial design was employed using choline levels of 0, 0.01, 0.04 and 0.16 mM, and methionine levels of 0, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8 and 1.6 mM. The 0.8 mM methionine level was best for all levels of choline, but because of the continued increase in total protein with each increase in choline, the choline requirement was uncertain. Experiment 2 was designed to more clearly establish the choline requirement. An 8 x 2 factorial design was used consisting of choline levels of 0, 0.01, 0.04, 0.1, 0.2, 0.4, 0.8 and 1.0 mM, and methionine levels of 0 and 0.8 mM. A choline level of 0.2 mM was found to be optimal. A third experiment was then performed to try to obtain biochemical evidence for a sparing effect of choline on the methionine requirement. A 4 x 5 factorial design using choline levels of 0, 0.1, 0.2, and 0.4 mM, methionine levels of 0, 0.4, 0.6, 0.8 and 1.2 mM, and labelled choline and methionine was employed. The 0.4 mM choline level resulted in a numerical but not significantly different increase in the amount of total protein measured at 0.4 and 0.6 mM methionine. When labelled methionine was included in the media, an increase in the amount of label incorporated into protein was observed with 0.4 mM choline at all levels of methionine. This label was subsequently shown to reside in the methionine residues. When methyl-labelled choline was included in the media, an increase in label incorporation into protein was also observed with 0.4 mM choline at all levels of methionine. Analysis showed that the label was not only in methionine residues, but also in a variety of other residues believed to be methylated derivatives of common amino acids. The results provide direct evidence for the proposed sparing effect of choline on the methionine requirement through a decrease in the amount of methionine utilized for transmethylation reactions and an increase in the amount of methionine available for protein synthesis.