Isolation of aminoacyl-tRNA and its labeling with stable-isotope tracers: Use in studies of human tissue protein synthesis.

摘要

We isolated aminoacyl-tRNA (60-70% yield) from human and rat tissues and measured, by GC/MS, its labeling in vivo by [15N]- and [13C]leucine. Tracer dilution artifacts seemed unlikely since, after infusion of L-[1-13C,15N]leucine into rats, (i) muscle leucyl-tRNA labeling exceeded tissue free leucine labeling, (ii) values were largely unaffected by storing over 5 min at 22 degrees C, and (iii) L-[2,4,5-methyl-13C]leucine was not incorporated into leucyl-tRNA during homogenization. Leucyl-tRNA labeling in liver and muscle suggested charging from extra- and intracellular pools: e.g., after infusing L-[1-13C,15N]leucine, rat muscle tissue free leucine 13C labeling (8.97 +/- 0.30 atom % excess) exceeded that by 15N (3.37 +/- 0.33 atom % excess), and both were significantly lower (P less than 0.02) than venous plasma (13C, 12.1 +/- 1.8; 15N, 5.54 +/- 0.6 atom % excess) indicating tracer dilution by transamination and by proteolysis; however, leucyl-tRNA labeling by either isotope (13C, 10.26 +/- 0.50; 15N, 4.72 +/- 0.72 atom % excess) was significantly above mixed tissue free leucine (P less than 0.05). Labeling of leucyl-tRNA in human erector spinae muscle (obtained after preoperative L-[1-13C]leucine infusion) was, at 4.98 +/- 0.43 atom % excess, lower (27%) than venous plasma leucine (P less than 0.05) and intermediate between muscle free leucine (9% lower; P less than 0.01) and venous alpha-ketoisocaproate (11% higher; P less than 0.02). Human placental leucyl-tRNA labeling (after predelivery tracer infusion) was 37% lower (P less than 0.05) than maternal uterine vein labeling but not significantly different from placental free leucine or umbilical arterial leucine.