Initial studies of the enzymatic composition of the surface membranes of the human pathogen Leishmania donovani and other trypanosomatids have identified a very active 3'-nucleotidase. Further studies of this enzyme have revealed several significant characteristics: 1) This ectoenzyme has a dual function as both a nucleotidase and nuclease which makes it ideally suited for initiating purine salvage from exogenous nucleic acids, in these organisms which lack the ability to synthesize the purine ring de novo. 2) The enzyme activity in L. donovani and in the insect trypanosomatid Crithidia luciliae is regulated by the supply of available purines in culture media, i.e. purine-starved parasites exhibit much higher levels of enzyme activity than do cells growing in complete medium. 3) The enzyme activity may be renatured following electrophoresis in SDS-polyacrylamide gels and catalytically active polypeptides can be identified. These features warrant further investigation. The studies proposed are intended to further the kinetic characterization of the phosphomonoesterase and phosphodiesterase activities of the 3'-nucleotidase/nuclease from L. donovani and from purine-starved C. luciliae. The enzyme will also be characterized with respect to its physicochemical properties. Another major goal of this proposal is to understand the mechanism of regulation of the 3'-nucleotidase/ nuclease, especially the regulated increase in the levels of enzyme activity when the protozoa are starved for purines. Our investigation of this trypanosomatid enzyme and its regulation will require the purification of the enzyme and development of specific antibody reagents. Recombinant DNA techniques, especially cDNA cloning of the gene, will be employed in order to address the issues indicated above. Results of such studies will provide new information on the uniqueness of this enzyme and its role in parasite survival. Studies of the enzyme's regulation will enable us to comprehend differential enzyme, and gene, expression in these parasites.
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