A Novel Stimulatory Action of Phosphorylcholine Plus Cytidine 5'-Triphosphate on Protein Synthesis in a Cell-free System

Abstract

Abstract In rat liver slices, phosphorylcholine stimulates incorporation of radioactive leucine into the proteins recovered from microsomes and medium. In a cell-free microsomal system, prepared from rat liver, additions of phosphorylcholine plus cytidine 5'-triphosphate increased dramatically the incorporation of radioactive leucine or phenylalanine into hot trichloracetic acid-insoluble proteins. Such increased protein synthesis was evident even when the usual requirements of ATP, guanidine 5'-triphosphate, or pH 5 enzyme for protein synthesis were considerably reduced. Phosphorylcholine plus CTP did not stimulate radioactive leucine incorporation into leucyl-tRNA as measured by subtracting the radioactivity incorporated into heated trichloracetic acid-insoluble proteins from corresponding unheated samples. Incorporation of labeled phenylalanine from phenylalanyl-tRNA into microsomal proteins was increased by phosphorylcholine plus CTP. Cycloheximide and puromycin, at a concentration of 10-4 m, abolished the activation effect of phosphorylcholine plus CTP on protein synthesis. Microsomal proteins were solubilized at the end of incubation by sodium deoxycholate treatment or by ultrasonic oscillation. The stimulatory effect of phosphorylcholine plus CTP on the specific activity of the soluble proteins was 2-fold higher than on that of the insoluble proteins of the pellet. Incubation in the presence of phospholipase C, brief ultrasonic oscillation of microsomes prior to incubation, and aging of microsomes in frozen storage caused a partial reduction or complete inhibition of the effect of phosphorylcholine plus CTP on protein synthesis. Incorporation of a tracer dose of phosphoryl[1,2-14C]choline, in the presence of phosphorylcholine, CTP, or CTP plus phosphorylcholine, into the lecithin fraction was extremely rapid in the incubation system normally used for studying protein synthesis. In stimulating protein synthesis, CTP could be replaced by uridine 5'-triphosphate, cytidine 5'-diphosphate, and cytidine 5'-phosphate, and these nucleotides, except UTP, also activated phosphorylcholine incorporation into lecithin. The different effect of UTP on phosphorylcholine incorporation into lecithin and on protein synthesis argues against a simple causal relationship between lecithin and protein synthesis. However, the results presented raise the possibility of a relationship between phosphorylcholine metabolism and protein synthesis.