USE OF O-18-LABELED LEUCINE AND PHENYLALANINE TO MEASURE PROTEIN-TURNOVER IN MUSCLE-CELL CULTURES AND POSSIBLE FUTILE CYCLING DURING AMINOACYLATION

Amino acids labelled with O-18 on both carboxy oxygen atoms have the p otential for use as non-recyclable tracers to measure protein turnover . During protein synthesis one of the labelled oxygen atoms is removed , and thus release of the mono-labelled amino acid could be used to de termine proteolysis. Primary cultures of embryonic-chick skeletal-musc le cells were used to test the use of O-18(2)-labelled Leu to measure proteolysis. For 9-day cultures, prelabelled on days 2-8 with medium c ontaining one-half the Leu as [O-18(2)]Leu and one-half as [H-2(3)]Leu , release of [O-18]Leu was less than 50 % that of [H-2]Leu over 24 h, suggesting a loss of the O-18 label by a mechanism other.than protein synthesis. Medium containing [O-18(2)]Leu, [H-2(3)]Leu, [O-18(2)]Phe a nd [C-13]Phe was then incubated with 9-day cultures to compare the rat e of loss of the O-18-label from Leu and Phe with the rate of uptake o f the non-carboxy-oxygen-labelled amino acids. Results for Leu demonst rated an 81% loss of the O-18 label compared with a 33% decrease in [H -2]Leu over 12 h. Loss of the O-18 label was four times as great for L eu as for Phe. Loss of the O-18 label was not decreased by addition of cycloheximide or by addition of a 3-fold excess of Ile, Val and Tyr; thus the loss of label was not due to protein synthesis alone or to mi sbinding to incorrect tRNAs. Infusion of the isotopes into pigs showed that the O-18 label of Leu was not lost during transamination to alph a-ketoisocaproate (alpha-oxoisohexanoate). The most probable explanati on is that the O-18 label is lost as a result of the enzymic deacylati on of tRNA, that this process is substantially faster for Leu than for Phe, and that this represents a potentially costly futile cycle for L eu.