Additional data suggestive of such a view are available from other types of experiments. Early investigations on nitrogen balance by Benedict, Folin, Gamble, Smith, and others point to the fact that the rate of protein catabolism varies with the dietary protein level. Since the protein level of the diet would be expected to exert a direct influence on synthesis rather than breakdown, the altered catabolic rate could well be caused by a change in the rate of synthesis.10 With the discovery of lysosomes in

eukaryotic cells it could be argued that energy was required for the transport of substrates into the lysosome Inhibitors,research,lifescience,medical or for maintenance of the low intralysosomal pH (see above), for example. The observation Inhibitors,research,lifescience,medical by Hershko and Tomkins that the activity of tyrosine aminotransferase

(TAT) was stabilized following depletion of ATP36 indicated that energy could be required at an early stage of the proteolytic process, most probably before proteolysis occurs. Yet, it did not provide a clue to the mechanism involved: energy could be used, for example, for specific modification of TAT, e.g. phosphorylation, that would sensitize it to degradation by the lysosome or by a yet unknown proteolytic mechanism, or for a modification that activates its putative protease. Inhibitors,research,lifescience,medical It could also be used for a more general lysosomal mechanism—one that involves transport of TAT into the Inhibitors,research,lifescience,medical lysosome, for example. The energy inhibitors inhibited

almost completely degradation of the entire population of cell proteins, confirming previous studies (e.g. Simpson10) and suggesting a general role for energy in protein catabolism. Yet, an interesting finding was that energy inhibitors had an effect that was distinct from that of protein synthesis inhibitors which affected only enhanced degradation (induced by steroid hormone depletion) but not basal degradation. This finding ruled out, at least partially, a tight linkage between protein synthesis and Inhibitors,research,lifescience,medical degradation. In bacteria, which lack lysosomes, an argument involving energy requirement for lysosomal degradation could not have been H 89 molecular weight proposed, but other indirect effects of ATP hydrolysis could have affected proteolysis in E. coli, such as phosphorylation of substrates and/or proteolytic enzymes, or maintenance of the “energized membrane state.” According to this model, proteins I BET 762 could become susceptible to proteolysis by changing their conformation, for example, following association with the cell membrane that maintains a local, energy-dependent gradient of a certain ion. While such an effect was ruled out,37 and since there was no evidence for a phosphorylation mechanism (although the proteolytic machinery in prokaryotes had not been identified at that time), it seemed that, at least in bacteria, energy was required directly for the proteolytic process.