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Leucine modulates protein translation in higher eukaryotes by affecting phosphorylation and the function of proteins that regulate the initiation and/or elongation steps. These include the initiation factor 4E binding protein 1 (4E-BP1), initiation factor 4E (eIF4E), initiation factor 2 (eIF2alpha), ribosomal S6 kinases (S6K1/2), and elongation factor 2 (eEF2). The alteration of protein translation by leucine starvation was studied during myogenic differentiation using the mouse C2C12 cell line as well as the role of rapamycin-sensitive mTOR (mammalian target of rapamycin) in the signaling of leucine in myotubes. A time course study showed that 1 h of leucine starvation decreased protein synthesis and S6K1 phosphorylation in myoblasts, whereas 3-5 h of starvation were necessary to induce such an alteration in myotubes. Although S6K1 phosphorylation was reduced in leucine-deprived myotubes, S6K2 and S6 phosphorylation were not affected. In contrast, rapamycin decreased the phosphorylation of S6K2 and S6 in myotubes. It is therefore likely that under the conditions present, the rapamycin-sensitive mTOR was not affected by leucine starvation. S6K1 dephosphorylation may thus be mTOR independent, and the functional mTOR/S6K2 pathway may maintain S6 phosphorylation. An increased phosphorylation of eEF2 in myoblasts and myotubes indicated that global protein synthesis was reduced via a decrease in translation elongation. An increased association between 4E-BP1 and eIF4E, and increased phosphorylation of eIF2alpha also contributed to decreasing protein synthesis in leucine-starved myoblasts. In contrast, in leucine-starved myotubes, there were no change in the 4E-BP1-eIF4E association or eIF2alpha phosphorylation, suggesting that these factors were not rate limiting for decreasing protein synthesis in leucine-deprived myotubes.

The effects of post-mortem storage and of a low-calcium-requiring neutral proteinase on the myofibrils of beef Longissimus dorsi and Rectus abdominis muscles and rabbit Longissirnus dorsi muscle were studied by measuring the Mg-Ca-enhanced myofibrillar ATPase activity and the changes in banding patterns on electrophoresis in sodium dodecyl sulphate. During ageing, the changes in the ATPase activity and myofibrillar proteins were qualitatively different between rabbit and bovine muscles, whilst differences in intensity were only observed between the two bovine muscles. The most prevalent component appearing upon conditioning had a mol. wt of 27 000 in rabbit muscle and of 30 000 in bovine muscles. In both species, the incubation of myofibrils with the low-calcium-requiring neutral proteinase mimicked the post-mortem changes. This would suggest that this enzyme has a qualitatively different effect on rabbit and bovine myofibrils.

Cathepsin L was purified from chicken liver lysosomes by a two-step procedure. Cathepsin L exhibited a single band of Mr 27,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, presented a high affinity for the substrate Z-Phe-Arg-NMec, was very unstable at neutral pH, and was inhibited by Z-Phe-Phe-CHN2. The complete amino acid sequence of cathepsin L has been determined and consists of 215 residues. The sequence was deduced from analysis of peptides generated by enzymatic digestions and by chemical cleavage at methionyl bonds. Comparison of the amino acid sequence of cathepsin L with those of rat liver cathepsins B and H and papain demonstrates a striking homology among their primary structures.