We have recently discovered an eukaryotic enzyme that has the same mechanism of action as certain microbial toxins. The object of this proposal is to characterize the newly discovered cellular mono-(ADP-ribosyl) transferase and its product, ADP-ribosylated EF-2. In addition, an inhibitor of the cellular ADP-ribosyltransferase will be purified and characterized. The goals of this proposal are to study the interaction of the cellular ADP-ribosyltransferase, EF-2 and the inhibitor of the enzyme in a defined in vitro system and to demonstrate the activity of this enzyme system in vivo along with the consequence of its activity on protein synthesis.
The specific aims are: 1. Determine if the cellular ADP-ribosyltransferase has a ubiquitous distribution among three hamster tissue types and beef liver. 2. Purify and characterize the cellular ADP-ribosyltransferase from hamsters. 3. Further purify hamster EF-2 and further characterize the ADP-ribosylated EF-2 product of the cellular enzyme. 4. Purify and characterize the cytoplasmic inhibitor of the cellular ADP-ribosyl-transferase. Determine the mechanism of inhibition of the cellular ADP-ribosyl-transferase activity. 5. Demonstrate the presence of ADP-ribosylated EF-2 in vivo and determine the consequence of its modification on protein synthesis.
