The long term goal of this proposal is to elucidate the mechanism(s) by which cells degrade their intracellular proteins. Ubiquitin, a small protein whose sequence has been remarkably conserved during evolution, is implicated in ATP-dependent proteolysis within animal cells. We have purified ubiquitin-lysozyme conjugates from hemin-inhibited rabbit reticulocyte lysates, and we have shown that these conjugates are rapidly degraded upon return to uninhibited lysates. More important, the conjugates are substrates for a large, ATP-dependent protease that does not degrade free lysozyme molecules. Using traditional biochemical fractionation techniques, we will purify to homogeneity the ATP-dependent protease, characterize it and produce antibodies to it. These antibodies will, in turn, be useful reagents for discovering whether the ATP-dependent protease is involved in the degradation of most cellular proteins or only a subset. In addition, we will purify sufficient quantities of the ubiquitin activating enzymes to produce antibodies to them. Finally, we will continue to analyze the structure of ubiquitin-lysozyme conjugates in an attempt to discover why they are anomalously large.
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