The effect of guanosine diphosphate and triphosphate on the incorporation of labeled amino acids into proteins.

We have previously described an enzyme system from rat liver which catalyzes the incorporation of labeled amino acids into proteins under anaerobic conditions (2, 3). This system consists of two liver cell fractions, the soluble cell fraction and the microsomes, to which are added a labeled amino acid, adenosine triphosphate,l and an ATP-generating system, such as phosphocreatine and creatine kinase. The microsome fraction contains ribonucleoprotein particles which appear to be the site of formation of the labeled proteins (4). The soluble cell fraction contains enzymes which catalyze the carboxyl activation of specific amino acids by ATP (5, 6). Before attempting to separate the enzymes of the soluble cell fraction which are essential for the formation of labeled proteins, it was important to investigate the cofactor requirements of the system further. By a purification of the two liver cell fractions, it has now been found that guanosine dior triphosphate is required in addition to ATP. The requirement for ATP had been demonstrated in a system containing microsomes and a dialyzed soluble cell fraction (2). The addition of ATP to this partially dialyzed system restored the incorporation to the level in the crude system, but the possibility remained that the partially dialyzed system contained adequate amounts of other cofactors essential for amino acid incorporation. Of the various known cofactors which were added along with ATP, guanosine diphosphate was found to give a small increase in the labeling of the proteins. Our interest in testing GDP arose from the finding of Sanadi et al. (7) that it is the cofactor in the phosphorylation of ADP coupled to the breakdown of succinyl coenzyme A. To determine