J. Biol. Chem., Vol. 259, Issue 3, 1436-1440, Feb, 1984
Leucine pools in normal and dystrophic chicken skeletal muscle cells in culture
PA Schneible and RB Young
The specific radioactivity of [3H]Leu in the extracellular, intracellular,
and Leu-tRNA pools of normal (white leghorn) and dystrophic (line 307)
embryonic chick breast muscle cultures was analyzed as a function of
equilibration time and extracellular Leu concentration (0.05-5 mM). The
primary results were the following 1) [3H]Leu equilibrated to a constant
specific radioactivity in the intracellular and Leu-tRNA pools within 2 min
after addition to both normal and dystrophic cultures. 2) After
equilibration, the extracellular [3H] Leu specific radioactivity in
dystrophic cell culture medium was lower than that of medium exposed to
normal cells (especially at low Leu concentrations), probably because of
increased release of unlabeled Leu from the dystrophic cells as a result of
faster protein breakdown. Accordingly, the specific radioactivities in the
intracellular and the Leu-tRNA pools were also lower in dystrophic cells.
3) At 5 mM extracellular Leu, the specific radioactivity in the Leu-tRNA
pool was approximately 40% lower than the specific radioactivity in the
intracellular pool in both normal and dystrophic cells. Thus, high
concentrations of extracellular Leu cannot be used to "flood out"
reutilization of unlabeled Leu (released by protein degradation) during
protein synthesis. 4) At 5.0 mM extracellular Leu, the specific
radioactivity of [3H]Leu in the intracellular pool was comparable to that
in the extracellular pool in normal and dystrophic cells; however, the
specific radioactivity of Leu-tRNA (i.e. the immediate precursor to protein
synthesis) was only 55-65% of the extracellular specific radioactivity in
normal and dystrophic cells. In conclusion, reutilization of Leu from
protein degradation is higher in dystrophic muscle cell cultures than in
normal muscle cell cultures, and accurate rates of protein synthesis in
cell cultures can only be obtained if specific radioactivity of amino acid
in tRNA is measured.
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