A stabilized enzyme system for amino acid incorporation.

Previous studies (1) of glutamic acid metabolism suggested an investigation of the role of this amino acid and its thio esters (2, 3) in amino acid incorporation. The studies by Zamecnik et al. (4, 5) on amino acid incorporation into freshly prepared rat liver microsomes in the presence of the soluble cell fraction and an ATPl-regenerating system appeared to provide a suitable test system for our purposes. However, considerable thioesterase activity was found in the microsomal preparations and still more in the soluble cell fraction (6), a fact which made a meaningful study of the utilization of glutamic acid thio esters in amino acid incorporation not feasible. It was then attempted to investigate the rate of incorporation of glutamic acid into microsomal preparations. In the absence of cysteine the incorporation of glutamic acid was of the order of magnitude of that of CY4-leucine, whereas in the presence of cysteine considerably more radioactivity was found in the proteins. This latter activity could be removed by treating the proteins with either SH compounds, such as excess cysteine and thioglycolic acid, or with performic acid. These observations suggested that glutamylcysteine (or GSH) formed during incubation was attached to the proteins by S-S linkages and was removed by the subsequent treatment.2 These findings are reminiscent of the observations that U4-glycine incorporated into proteins of partially fractionated liver