Microtechnique for Determining the Specific Activity of Radioactive Intracellular Leucine and Applications to in Vivo Studies of Protein Synthesis

A microtechnique has been developed for the determination of specific activities of radioactive leucine in the nanogram range, with an accuracy of at least 7%. The technique depends on the coupling of [3H]leucine to [14C]2,4-dinitrofluorobenzene. By use of inulin in the incubation medium, a correction can be made for extracellular leucine contaminating the tissue, and thus the specific activity of the intracellular leucine pool can be estimated. The technique has been applied to determining rates of protein synthesis in a protein-secreting gland, the galea of the silkmoth, Antheraea polyphemus, after progressive exposure to actinomycin D. Actinomycin D is shown to superinduce incorporation of [3H]leucine in the differentiation-specific product of the galea, the zymogen of cocoonase. However, superinduction is illusory; when changes in specific activity of the intracellular pool are taken into account, no actinomycin effect on zymogen synthesis is apparent.